1: Br J Cancer. 2005 Oct 31;93(9):1019-23. Nuclear localisation of nuclear factor-kappaB transcription factors in prostate cancer: an immunohistochemical study. Lessard L, Begin LR, Gleave ME, Mes-Masson AM, Saad F. 1Centre de recherche du CHUM, and Institut du cancer de Montreal, 1560 Sherbrooke Est, Montreal, Quebec, Canada H2L 4M1. Several reports suggest that the canonical nuclear factor-kappaB (NF-kappaB) pathway is constitutively activated in a subset of prostate cancer cells. However, except for RelA (p65), little is known about the status of NF-kappaB transcription factors in prostate cancer tissues. To clarify the status of NF-kappaB subunits, we analysed the expression and subcellular localisation of RelA, RelB, c-Rel, p50, and p52 on tissue array sections containing respectively 344, 346, 369, 343, and 344 cores from 75 patients. The subcellular localisation of NF-kappaB factors was tested against relevant clinical parameters (preoperative prostate-specific antigen, pathological stage, and postoperative Gleason grade). With the exception of c-Rel, each subunit was detected in the nucleus of cancer cells: significant nuclear expression of RelB, RelA, p52, and p50 was seen in 26.6, 15.6, 10.7, and 10.5% of cores, respectively. Surprisingly, cores expressing both nuclear RelA and p50 canonical pathway proteins were less frequently observed than cores expressing other subunit combinations such as RelB-p52 and RelA-RelB. In addition, the nuclear localisation of RelB correlated with patient's Gleason scores (Spearman correlation: 0.167; P=0.018). The nuclear localisation of both canonical and noncanonical NF-kappaB subunits in prostate cancer cells suggests for the first time that different NF-kappaB pathways and dimers may be activated in the progression of the disease.British Journal of Cancer (2005) 93, 1019-1023. doi:10.1038/sj.bjc.6602796 www.bjcancer.com Published online 4 October 2005. PMID: 16205698 [PubMed - in process] --------------------------------------------------------------- 2: Am J Physiol Renal Physiol. 2005 Oct 4; [Epub ahead of print] Persistent NF-{kappa}B activation in renal epithelial cells in mouse modelof HIV-associated nephropathy. Martinka S, Bruggeman LA. Department of Medicine and Rammelkamp Center for Education and Research, Case Western Reserve University School of Medicine, Cleveland, OH, USA. HIV-associated nephropathy is caused, in part, by direct infection of kidney epithelial cells by HIV-1. In the spectrum of pathogenic host-virus interactions, abnormal activation or suppression of host transcription factors is common. NF-kappaB is a necessary host transcription factor for HIV-1 gene expression, and it has been shown that NF-kappaB activity is dysregulated in many naturally infected cell types. We show here that renal glomerular epithelial cells (podocytes) expressing the HIV-1 genome, similar to infected immune cells, also have a dysregulated and persistent activation of NF-kappaB. Although podocytes produce p50, p52, RelA, RelB, and c-Rel; electrophoretic mobility shift assays and immunocytochemistry showed a predominant nuclear accumulation of p50/RelA-containing NF-kappaB dimers in HIV-1 expressing podocytes as compared to normal. In addition, the expression level of a transfected NF-kappaB reporter plasmid was significantly higher in HIVAN podocytes. The mechanism of NF-kappaB activation involved increased phosphorylation of IkappaBalpha, resulting in an enhanced turnover of the IkappaBalpha protein. There was no evidence for regulation by IkappaBbeta or the alternate pathway of NF- kappaB activation. Altered activation of this key host transcription factor likely plays a role in the well described cellular phenotypic changes observed in HIVAN such as proliferation. Studies with inhibitors of proliferation and NF-kappaB suggest that NF-kappaB activation may contribute to the proliferative mechanism in HIVAN. In addition, since NF-kappaB regulates many aspects of inflammation, this dysregulation may also contribute to disease severity and progression through regulation of pro-inflammatory processes in the kidney microenvironment. PMID: 16204413 [PubMed - as supplied by publisher] --------------------------------------------------------------- 3: Neurochem Int. 2005 Dec;47(8):545-55. Epub 2005 Sep 22. Distinct nuclear factor-kappaB/Rel proteins have opposing modulatory effects in glutamate-induced cell death in HT22 cells. Ishige K, Tanaka M, Arakawa M, Saito H, Ito Y. Department of Pharmacology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan. Members of the nuclear factor-kappaB (NF-kappaB)/Rel family (p50, p52, p65 (RelA), RelB and c-Rel) is sequestered in the cytoplasm through its tight association with the inhibitor of NF-kappaB (IkappaB). NF-kappaB has been shown to function as key regulators of either cell death or survival in neurons after activation of the cells by various extracellular signals. In the study presented here, we investigated whether the selective activation of diverse NF-kappaB/Rel family members in HT22 cells might lead to distinct effects on glutamate-induced cell death. Exposing HT22 cells to glutamate, which blocks cystine uptake into the cells via inhibition of the glutamate-cystine antiporter, resulted in a transient activation of IkappaB and NF-kappaB/Rel and caused delayed cell death. Aspirin, which has been shown to block phosphorylation of the IkappaB component of the cytoplasmic NF-kappaB complex, significantly suppressed glutamate-induced cell death, whereas the NF-kappaB decoy oligonucleotide potentiated it. The inhibition of NF-kappaB/Rel protein expression by antisense oligonucleotides showed that p65 is involved in glutamate-mediated cell death, whereas p50 is involved in inhibitory pathways of the cell death. These findings suggest that in HT22 cells, the balance between promoting and presenting cell death to glutamate-induced oxidative stress relies on the activation of distinct NF-kappaB proteins. PMID: 16183169 [PubMed - in process] --------------------------------------------------------------- 4: Genes Dev. 2005 Sep 15;19(18):2138-51. A c-Rel subdomain responsible for enhanced DNA-binding affinity and selective gene activation. Sanjabi S, Williams KJ, Saccani S, Zhou L, Hoffmann A, Ghosh G, Gerondakis S, Natoli G, Smale ST. Howard Hughes Medical Institute, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095-1662, USA. The NF-kappaB family members p65 (RelA) and c-Rel recognize similar DNA sequences, yet the phenotypes of mutant mice suggest that these proteins regulate distinct sets of genes. Here we demonstrate that 46 unique residues within an 86-residue segment of the Rel homology region (RHR) of c-Rel are responsible for the c-Rel requirement for Il12b gene induction by lipopolysaccharide in bone marrow-derived macrophages. These same residues were responsible for the c-Rel requirement for Il12a induction in dendritic cells, and in both instances, no evidence of c-Rel-specific coactivator interactions was found. Although the residues of c-Rel and p65 that contact specific bases and the DNA backbone within nuclear factor-kappaB (NF-kappaB) recognition sequences are identical, homodimers of c-Rel and of a chimeric p65 protein containing the critical c-Rel residues bound with high affinity to a broader range of NF-kappaB recognition sequences than did wild-type p65 homodimers. These results demonstrate that the unique functions of closely related transcription factor family members can be dictated by differences in the range of DNA sequences recognized at high affinity, despite having similar binding site consensus sequences and DNA contact residues. PMID: 16166378 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Int J Cancer. 2005 Aug 16; [Epub ahead of print] Ursodeoxycholic acid inhibits interleukin beta 1 and deoxycholic acid-induced activation of NF-kappaB and AP-1 in human colon cancer cells. Shah SA, Volkov Y, Arfin Q, Abdel-Latif MM, Kelleher D. Department of Clinical Medicine and Dublin Molecular Medicine Centre, Trinity Centre for Health Sciences, St. James's Hospital, Dublin, Ireland. Deoxycholic acid (DCA) has been implicated in colorectal carcinogenesis in humans with effects on proliferation and apoptosis, mediated at least in part by activation of transcription factors nuclear factor kappa B (NF-kappaB), activator protein 1 (AP-1) and protein kinase C (PKC) enzymes. Ursodeoxycholic acid (UDCA) is reported to reduce the frequency of colonic carcinogenesis in ulcerative colitis patients. Hence, we postulated that it might differ from DCA in its regulation of these transcription factors. The aim of the study was to determine effects of DCA and UDCA on NF-kappaB and AP-1 activation and explore its relationship to PKC. Human colonic tumour cell lines HCT116 were treated with DCA, UDCA, alone or pretreated with UDCA followed by DCA or IL-1beta. In other experiments, cells were pretreated with PKC inhibitors and then stimulated with DCA and IL-1beta or PMA. Gel shift assays were performed on nuclear extracts of the cells for NF-kappaB and AP-1 analysis. Western blot analyses and immunofluorescence were performed for Rel A (p65) and IkappaB-alpha levels on the treated cells. DCA increased NF-kappaB and AP-1 DNA binding. UDCA did not increase DNA binding of NF-kappaB and AP-1 and UDCA pretreatment inhibited DCA-induced NF-kappaB and AP-1 DNA binding. PKC inhibitors blocked DCA-induced NF-kappaB and AP-1 activation. These results were validated by Western blot analysis for RelA and IkappaB-alpha. In conclusion, UDCA did not induce NF-kappaB and AP-1 DNA binding but also blocked DCA-induced NF-kappaB and AP-1 activation. These findings suggest a possible mechanistic role for UDCA in blocking pathways thought to be involved in colon carcinogenesis. (c) 2005 Wiley-Liss, Inc. PMID: 16106402 [PubMed - as supplied by publisher] --------------------------------------------------------------- 6: Infect Immun. 2005 Aug;73(8):4972-81. Borrelia burgdorferi rel is responsible for generation of guanosine-3'-diphosphate-5'-triphosphate and growth control. Bugrysheva JV, Bryksin AV, Godfrey HP, Cabello FC. Department of Microbiology and Immunology, Basic Science Building, New York Medical College, Valhalla, NY 10595, USA. The global transcriptional regulator (p)ppGpp (guanosine-3'-diphosphate-5'-triphosphate and guanosine-3',5'-bisphosphate, collectively) produced by the relA and spoT genes in Escherichia coli allows bacteria to adapt to different environmental stresses. The genome of Borrelia burgdorferi encodes a single chromosomal rel gene (BB0198) (B. burgdorferi rel [rel(Bbu)]) homologous to relA and spoT of E. coli. Its role in (p)ppGpp synthesis, bacterial growth, and modulation of gene expression has not been studied in detail. We constructed a rel(Bbu) deletion mutant in an infectious B. burgdorferi 297 strain and isolated an extrachromosomally complemented derivative of this mutant. The mutant did not synthesize rel(Bbu) mRNA, Rel(Bbu) protein, or (p)ppGpp. This synthesis was restored in the complemented derivative, confirming that rel(Bbu) is necessary and sufficient for (p)ppGpp synthesis and degradation in B. burgdorferi. The rel(Bbu) mutant grew well during log phase in complete BSK-H but reached lower cell concentrations in the stationary phase than the wild-type parent, suggesting that (p)ppGpp may be an important factor in the ability of B. burgdorferi to adapt to stationary phase. Deletion of rel(Bbu) did not eliminate the temperature-elicited OspC shift, nor did it alter bmp gene expression or B. burgdorferi antibiotic susceptibility. Although deletion of rel(Bbu) eliminated B. burgdorferi virulence for mice, which was not restored by complementation, we suggest that rel(Bbu)-dependent accumulation of (p)ppGpp may be important for in vivo survival of this pathogen. PMID: 16041012 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Immunol. 2005 Aug 1;175(3):1834-42. Constitutive nuclear expression of the I kappa B kinase complex and its activation in human neutrophils. Ear T, Cloutier A, McDonald PP. Pulmonary Division, Faculty of Medicine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada. A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-kappaB/Rel proteins and IkappaB-alpha in the nucleus. In this study, we show that in these cells, IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma also partially localize to the nucleus, whereas IKK-related kinases (IKKepsilon, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-kappaB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKbeta and IKKgamma become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKalpha transiently vanishes from both compartments in what appears to be an IKKbeta-dependent process. These responses are paralleled by the degradation of IkappaB-alpha, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IkappaB-alpha phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (alpha, beta, gamma) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-kappaB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IkappaB signaling cascade within the cell nucleus. PMID: 16034126 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Oncogene. 2005 Sep 29;24(43):6574-83. Immortalized fibroblasts from NF-kappaB RelA knockout mice show phenotypic heterogeneity and maintain increased sensitivity to tumor necrosis factor alpha after transformation by v-Ras. Gapuzan ME, Schmah O, Pollock AD, Hoffmann A, Gilmore TD. Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA. Activation of the NF-kappaB pathway can either promote or block apoptosis and oncogenesis in different cell types and circumstances. In this report, we show that independently derived immortalized mouse embryonic fibroblast cell lines prepared from RelA knockout mice have different phenotypes, based on their sensitivity to tumor necrosis factor alpha (TNFalpha)-induced apoptosis, morphology, ability to form colonies in soft agar, and the presence of distinct kappaB site-binding complexes. In addition, these RelA-deficient cell lines appear to have distinct alterations in the p53 pathway, which correlate with the normal vs transformed status of individual cell lines. We have also infected mouse embryonic fibroblasts lacking RelA, c-Rel or p50 with a retrovirus for the expression of v-Ha-Ras to determine whether individual NF-kappaB family members are required for Ras-mediated transformation. All three NF-kappaB-deficient cell types could be transformed by v-Ha-Ras. However, v-Ras-infected RelA-deficient cells formed colonies in soft agar at an approximately fourfold reduced efficiency compared to v-Ras-transformed control mouse 3T3 and p50-deficient cells. Ras transformation did not alter the sensitivity of RelA-deficient cells to TNFalpha-induced apoptosis, and Ras transformation did not affect the general resistance of 3T3, c-Rel-deficient, and p50-deficient cells to TNFalpha-induced apoptosis. However, TNFalpha specifically and dose-dependently decreased the ability of v-Ras-transformed RelA-deficient cells to form colonies in soft agar. These results suggest that RelA is a potential protein target for human tumors driven by oncogenic Ras mutations, but caution that inhibition of RelA may promote tumorigenesis in some circumstances. PMID: 16027734 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Oncogene. 2005 Sep 29;24(43):6482-91. Activation of NF-kappaB following detachment delays apoptosis in intestinal epithelial cells. Yan SR, Joseph RR, Rosen K, Reginato MJ, Jackson A, Allaire N, Brugge JS, Jobin C, Stadnyk AW. Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3H4R2. We reported earlier that IL-1beta, an NF-kappaB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-kappaB-regulated products and therefore aimed to determine whether NF-kappaB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IkappaBalpha becomes phosphorylated immediately following detachment and that levels of phospho-IkappaBalpha peaked within 20 min. Phosphorylation of IkappaBalpha was followed by Rel A (p65) nuclear translocation. Increased NF-kappaB activity following detachment was confirmed using the detection of NF-kappaB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IkappaBalpha protein and pharmacological inhibitors of NF-kappaB resulted in the failure to phosphorylate IkappaBalpha, a more rapid activation of caspases and earlier apoptosis. We also detected that IkappaB kinase alpha (IKKalpha) and not IKKbeta became phosphorylated following detachment. Since IKKalpha is activated by NF-kappaB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IkappaBalpha and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-kappaB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells. PMID: 16007176 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Biol Chem. 2005 Aug 12;280(32):29256-62. Epub 2005 Jun 17. Induction of NR4A orphan nuclear receptor expression in macrophages in response to inflammatory stimuli. Pei L, Castrillo A, Chen M, Hoffmann A, Tontonoz P. Howard Hughes Medical Institute and Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095, USA. Oxidized lipids and inflammatory cytokines are believed to play a causal role in atherosclerosis through the regulation of gene expression in macrophages and other cells. Previous work has implicated the nuclear receptors peroxisome proliferator-activated receptor and liver X receptor in the control of lipid-dependent gene expression and inflammation. Here we demonstrate that expression of a third group of nuclear receptors, the NR4A ligand-independent orphan receptors, is highly inducible in macrophages by diverse inflammatory stimuli. Treatment of macrophages with lipopolysaccharide (LPS), cytokines, or oxidized lipids triggers the transcriptional induction of Nur77 (NR4A1), Nurr1 (NR4A2), and NOR1 (NR4A3) expression. Several lines of evidence point to the NF-kappaB signaling pathway as a principal mediator of inducible NR4A expression in macrophages. Analysis of the murine and human Nur77 promoters revealed two highly conserved NF-kappaB response elements. Mutation of these elements inhibited LPS-dependent expression of the Nur77 promoter in transient transfection assays. Furthermore, induction of Nur77 expression by LPS was severely compromised in fibroblasts lacking the three NF-kappaB subunits, Nfkb1, c-Rel, and RelA. Consistent with its ability to be induced by oxidized lipids, Nur77 was expressed in macrophages within human atherosclerotic lesions. These results identified NR4A nuclear receptors as potential transcriptional mediators of inflammatory signals in activated macrophages. PMID: 15964844 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Biochem Biophys Res Commun. 2005 Aug 5;333(3):896-907. A cell line model for the differentiation of human dendritic cells. Berges C, Naujokat C, Tinapp S, Wieczorek H, Hoh A, Sadeghi M, Opelz G, Daniel V. Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany. We have identified human monocytic (THP-1) and myelogenous CD34+ (KG-1) leukemia cell lines that can be differentiated rapidly into mature dendritic cells (DCs) when cultured in serum-free medium containing GM-CSF, TNF-alpha, and ionomycin. These hematopoietic cell line-derived DCs are highly pure and monotypic, and display the morphologic, phenotypic, molecular, and functional properties of DCs generated from human donor-derived monocytes or CD34+ hematopoietic progenitor cells. During differentiation into mature DCs, the cells exhibit de novo cell-surface expression of CD83, CD80, CD86, CD40, CD206, CD209, CD120a, CD120b, and intracellular synthesis of IL-10, increase their endocytotic capacity, and acquire characteristic stellate morphology. To further define the cells as DCs, cytosolic induction and upregulation of RelB and RelA (p65), transcription factors of the NF-kappaB/Rel family essential for differentiation and maturation of DCs, as well as upregulation of the immunoproteasome subunits LMP2, LMP7, and MECL-1, and the proteasome activator PA28alpha, components essential for efficient MHC class I peptide antigen processing, were demonstrated during differentiation of the cells. In contrast to the cell lines, the cell line-derived mature DCs are capable of stimulating allogeneic CD4+ and CD8+ T cells, ultimately defining them as potent antigen-presenting cells. The approach to differentiate THP-1 and KG-1 cells into immature and mature DCs may serve as an experimental model to study molecular events and pathways that govern the differentiation of human malignant myeloid precursors, monocytes, and CD34+ hematopoietic progenitor cells into DCs. PMID: 15963458 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Invest Dermatol. 2005 Jun;124(6):1275-83. Differential expression of phosphorylated NF-kappaB/RelA in normal and psoriatic epidermis and downregulation of NF-kappaB in response to treatment with etanercept. Lizzul PF, Aphale A, Malaviya R, Sun Y, Masud S, Dombrovskiy V, Gottlieb AB. Clinical Research Center, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901-0019, USA. lizzulpa@umdnj.edu Etanercept, a recombinant human tumor necrosis factor (TNF) receptor fusion protein, is FDA approved for psoriasis and psoriatic arthritis. TNFalpha increases the synthesis of proinflammatory cytokines and leads to the activation of multiple signaling pathways, including nuclear factor kappa B (NF-kappaB). The Rel/NF-kappaB transcription factors play a central role in numerous cellular processes, including the stress response and keratinocyte proliferation and differentiation. Utilizing a phosphorylation-specific antibody, we examined the expression of active nuclear NF-kappaB/RelA via immunohistochemistry in normal skin, non-lesional psoriatic skin, lesional psoriatic skin, and lesional skin from patients treated with etanercept. There was no expression of active nuclear NF-kappaB in the normal epidermis, whereas a basal level of constitutive active phosphorylated NF-kappaB/RelA was present in uninvolved epidermis from psoriasis patients. There was also significant upregulation of active phosphorylated NF-kappaB/RelA in the epidermis from psoriatic plaques. Serial biopsies from psoriasis patients treated with etanercept at 1, 3, and 6 mo demonstrated a significant downregulation of phosphorylated NF-kappaB/RelA, which correlated with decreases in epidermal thickness, restoration of normal markers of keratinocyte differentiation, and clinical outcomes. These data suggest that activation of NF-kappaB plays a significant role in the pathogenesis of psoriasis and that a potential mechanism of action for TNF-targeting agents is downregulation of NF-kappaB transcriptional activity. Publication Types: Clinical Trial Controlled Clinical Trial PMID: 15955104 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Biochem J. 2005 Aug 15;390(Pt 1):317-24. Activation of RelA homodimers by tumour necrosis factor alpha: a possible transcriptional activator in human vascular endothelial cells. Marui N, Medford RM, Ahmad M. Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA. In vascular endothelial cells, cytokines induce genes that are expressed in inflammatory lesions partly through the activation of transcription factor NF-kappaB (nuclear factor-kappaB). Among the members of the NF-kappaB/rel protein family, homodimers of the RelA subunit of NF-kappaB can also function as strong transactivators when expressed in cells. However, the functional role of endogenous RelA homodimers has not been clearly elucidated. We investigated whether RelA homodimers are induced in cytokine-treated vascular endothelial cells. Gel mobility-shift and supershift assays revealed that a cytokine TNFalpha (tumour necrosis factor alpha) activated both NF-kappaB1/RelA heterodimers and RelA homodimers that bound to a canonical kappaB sequence, IgkappaB (immunoglobulin kappaB), in SV40 (simian virus 40) immortalized HMEC-1 (human dermal microvascular endothelial cell line 1). In HMEC-1 and HUVEC (human umbilical-vein endothelial cells), TNFalpha also induced RelA homodimers that bound to the sequence 65-2kappaB, which specifically binds to RelA homodimers but not to NF-kappaB1/RelA heterodimers in vitro. Deoxycholic acid, a detergent that can dissociate the NF-kappaB-IkappaB complex (where IkappaB stands for inhibitory kappaB), induced the binding of the RelA homodimers to 65-2kappaB from the cytosolic fraction of resting HMEC-1. Furthermore, TNFalpha induced the transcriptional activity of a reporter gene that was driven by 65-2kappaB in HMEC-1. These results suggest that in addition to NF-kappaB1/RelA heterodimers, TNFalpha also induces RelA homodimers that are functionally active. Thus RelA homodimers may actively participate in cytokine regulation of gene expression in human vascular endothelial cells. PMID: 15876188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Nature. 2005 Apr 28;434(7037):1138-43. IKKalpha limits macrophage NF-kappaB activation and contributes to the resolution of inflammation. Lawrence T, Bebien M, Liu GY, Nizet V, Karin M. Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California San Diego, 9500 Gilman Drive, California 92093, USA. t.lawrence@imperial.ac.uk Inflammation and innate immunity involve signalling pathways leading to the production of inflammatory mediators. Usually such responses are self-limiting, but aberrant resolution of inflammation results in chronic diseases. Much attention has focused on pro-inflammatory signalling but little is known about the mechanisms that resolve inflammation. The IkappaB kinase (IKK) complex contains two catalytic subunits, IKKalpha and IKKbeta, and controls the activation of NF-kappaB transcription factors, which play a pivotal role in inflammation. Ample evidence indicates that IKKbeta mediates NF-kappaB activation in response to pro-inflammatory cytokines and microbial products. IKKalpha regulates an alternative pathway important for lymphoid organogenesis, but the role of IKKalpha in inflammation is unknown. Here we describe a new role for IKKalpha in the negative regulation of macrophage activation and inflammation. IKKalpha contributes to suppression of NF-kappaB activity by accelerating both the turnover of the NF-kappaB subunits RelA and c-Rel, and their removal from pro-inflammatory gene promoters. Inactivation of IKKalpha in mice enhances inflammation and bacterial clearance. Hence, the two IKK catalytic subunits have evolved opposing but complimentary roles needed for the intricate control of inflammation and innate immunity. PMID: 15858576 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Mol Cell Biol. 2005 Apr;25(8):2957-68. 17beta-estradiol inhibits inflammatory gene expression by controlling NF-kappaB intracellular localization. Ghisletti S, Meda C, Maggi A, Vegeto E. Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. Estrogen is an immunoregulatory agent, in that hormone deprivation increases while 17beta-estradiol (E2) administration blocks the inflammatory response; however, the underlying mechanism is still unknown. The transcription factor p65/relA, a member of the nuclear factor kappaB (NF-kappaB) family, plays a major role in inflammation and drives the expression of proinflammatory mediators. Here we report a novel mechanism of action of E2 in inflammation. We observe that in macrophages E2 blocks lipopolysaccharide-induced DNA binding and transcriptional activity of p65 by preventing its nuclear translocation. This effect is selectively activated in macrophages to prevent p65 activation by inflammatory agents and extends to other members of the NF-kappaB family, including c-Rel and p50. We observe that E2 activates a rapid and persistent response that involves the activation of phosphatidylinositol 3-kinase, without requiring de novo protein synthesis or modifying Ikappa-Balpha degradation and mitogen-activated protein kinase activation. Using a time course experiment and the microtubule-disrupting agent nocodazole, we observe that the hormone inhibits p65 intracellular transport to the nucleus. This activity is selectively mediated by estrogen receptor alpha (ERalpha) and not ERbeta and is not shared by conventional anti-inflammatory drugs. These results unravel a novel and unique mechanism for E2 anti-inflammatory activity, which may be useful for identifying more selective ligands for the prevention of the inflammatory response. PMID: 15798185 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Blood. 2005 Jul 1;106(1):235-40. Epub 2005 Mar 24. Azidothymidine inhibits NF-kappaB and induces Epstein-Barr virus gene expression in Burkitt lymphoma. Kurokawa M, Ghosh SK, Ramos JC, Mian AM, Toomey NL, Cabral L, Whitby D, Barber GN, Dittmer DP, Harrington WJ Jr. Department of Dermatology, Medical College, University of Miyazaki, Japan. The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL. PMID: 15790788 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: J Bacteriol. 2005 Apr;187(7):2439-47. The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression. Dahl JL, Arora K, Boshoff HI, Whiteford DC, Pacheco SA, Walsh OJ, Lau-Bonilla D, Davis WB, Garza AG. School of Molecular Biosciences, Washington State University, Science Hall, Room 301, Pullman, WA 99164, USA. johndahl@wsu.edu The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel(Msm). The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure. PMID: 15774887 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Tsitologiia. 2004;46(12):1064-72. [alpha-Actinin-4 and p65/RelA subunit of NF-kappaB transcription factor are co-localized and migrate together into the nucleus in EGF-stimulated A431 cell] [Article in Russian] Babakov VN, Bobkov DE, Petukhova OA, Turoverova LV, Kropacheva IV, Podol'skaia EP, Pinaev GP. The NF-kappaB/Rel family of transcription factors in mammalian cells regulates inducible transcription of a large number of genes in response to diverse stimuli. Despite a great number of publications on this subject, little is known about precise NF-kappaB localization in the cytoplasm. As previously demonstrated, in normal rat fibroblast and human epidermoid carcinoma A431 cells p65/RelA subunit of NF-kappaB is co-localized in the cytoplasm with actin structures. However, the mechanism of NF-kappaB interaction with actin remains unclear. We have investigated localization of p65/RelA subunit NFkappaB and alpha-actinin isoforms during cell activation by epidermal growth factor (EGF). Using confocal microscopy, we have shown that alpha-actinin-4 and p65/RelA subunit of NF-kappaB transcription factor are co-localized in A431 cells. Cell treatment with EGF leads to translocation of the proteins to membrane ruffles, and eventually to migration into the nucleus. Pretreatment of A431 cells with cytochalasin D or wortmannin prior to EGF treatment increases p65/RelA and alpha-actinin-4 accumulation in nuclear extracts. Co-localization of alpha-actinin-4 with p65/RelA subunit of NF-kappaB was found in nuclei isolated from stimulated cells. These results support the notion that actin cytoskeleton reorganization and alpha-actinin-4 are involved in NF-kappaB signaling. PMID: 15747836 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: J Biol Chem. 2005 Apr 29;280(17):17435-48. Epub 2005 Feb 18. Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling. Tian B, Nowak DE, Jamaluddin M, Wang S, Brasier AR. Department of Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1060, USA. Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway. PMID: 15722553 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Immunology. 2005 Mar;114(3):313-21. Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor. Rahim SS, Khan N, Boddupalli CS, Hasnain SE, Mukhopadhyay S. Centre for DNA Fingerprinting and Diagnostics (CDFD), Nacharam, Hyderabad, India. Interleukin-10 (IL-10) is known to inhibit IL-12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor-kappaB (NF-kappaB). We demonstrate that the c-rel transcription factor also plays a major role in IL-10-mediated IL-12 suppression. Treatment of macrophages with recombinant IL-10 inhibited nuclear c-rel levels, whereas addition of neutralizing anti-IL-10 antibody up-regulated both nuclear c-rel levels and IL-12 production by macrophages. Decreased nuclear c-rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IkappaBalpha) in the cytoplasm, indicating that IL-10 prevents degradation of IkappaBalpha and the subsequent translocation of c-rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c-rel at a concentration of 10 nm, when used with anti-IL-10 antibody, resulted in reduced expression of IL-12. In a complementary experiment, in vitro transient expression of p65 NF-kappaB could not rescue the inhibitory effect of IL-10 on IL-12 production, suggesting that NF-kappaB alone was not sufficient to restore IL-12 production during IL-10 treatment. However, over-expression of c-rel resulted in IL-12 restoration upon stimulation with lipopolysaccharide plus interferon-gamma during IL-10 treatment. Our studies highlight the involvement of c-rel in IL-10-mediated IL-12 regulation. PMID: 15720433 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Oncogene. 2005 Mar 3;24(10):1749-66. Role of NF-kappaB signaling in hepatocyte growth factor/scatter factor-mediated cell protection. Fan S, Gao M, Meng Q, Laterra JJ, Symons MH, Coniglio S, Pestell RG, Goldberg ID, Rosen EM. Department of Oncology, Lombardi Cancer Center/Georgetown University, 3970 Reservoir Road, NW, Box 571469, Washington, DC 20057-1469, USA. The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIalpha inhibitor). We investigated the role of nuclear factor kappa B (NF-kappaB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin-Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-kappaB associated with the transient loss of the inhibitory subunit IkappaB-alpha. Exposure to HGF/SF caused the activation of an NF-kappaB luciferase reporter that was blocked or attenuated by the expression of a mutant 'super-repressor' IkappaB-alpha. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-kappaB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-kappaB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IkappaB-alpha super-repressor. The ability of HGF/SF to activate NF-kappaB signaling was dependent on c-Akt --> Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3'-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-kappaB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-kappaB activity. HGF/SF induced the expression of several known NF-kappaB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-kappaB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-kappaB through a c-Akt --> Pak1 signaling pathway that is also dependent on Src, and that NF-kappaB contributes to HGF/SF-mediated protection against ADR. PMID: 15688034 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Exp Dermatol. 2005 Jan;14(1):17-25. Deficient translocation of c-Rel is associated with impaired Th1 cytokine production in T cells from atopic dermatitis patients. Dieckhoff K, Graf P, Beinhauer B, Schwaerzler C, Carballido JM, Neumann C, Zachmann K, Jung T. Department of Dermatology, University Goettingen, Germany. Decreased production of T helper type 1 (Th1) cytokines, such as interferon-gamma (IFN-gamma) or interleukin-2 (IL-2), is a hallmark of atopic diseases. While accessory signals from antigen-presenting cells may be missing, T cells themselves may be suppressed in their ability to produce substantial amounts of Th1 cytokines. We show, in this study, that T cell receptor (TCR)-activated T cells from atopic dermatitis (AD) patients proliferate less than control T cells and produce lower amounts of IFN-gamma and IL-2, but comparable amounts of IL-4. Because mice lacking the nuclear factor kappa B (NF-kappaB) transcription factors - p65 or c-Rel - show reduced Th1, but undisturbed Th2 responses, we investigated the role of c-Rel and p65 for Th1 cytokine production in T cells from healthy and severe AD patients. TCR-activated primary T cells from healthy donors treated with c-Rel antisense oligonucleotides produced lower levels of IL-2 and IFN-gamma and proliferated less efficiently than the corresponding control T cells. Moreover, transfection of primary T cells with c-Rel or p65 enhanced proliferation and production of IL-2 and IFN-gamma. Nuclear extracts of activated primary T cells from AD donors bound weakly to NF-kappaB-specific oligonucleotides, compared to extracts from healthy control T cells. Western blotting studies revealed that nuclear, but not cytosolic, extracts from T cells of AD patients lacked significant amounts of c-Rel and p65. T cell clones derived from AD patients failed to sufficiently translocate c-Rel and p65 into the nucleus following activation. Thus, impaired nuclear translocation of c-Rel and p65 may determine an impaired Th1 cytokine response in AD. PMID: 15660915 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Immunopharmacol Immunotoxicol. 2004;26(4):545-58. Suppression of nuclear translocation of nuclear factor-kappaB and nuclear factor of activated T cells by Younggaechulgam-tang. Shin HY, Song YS, Hwang CY, Shin TY, Kim HM. Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, Seoul, South Korea. Younggaechulgam-tang (YGCGT) is known to suppress inflammatory and autoimmune responses, and it has clinically been used among Oriental medical doctors in South Korea. We investigated YGCGT-mediated changes in downstream T cell signal transduction. The expression levels of nuclear factor-kappaB (NFkappaB) subunit RelA and nuclear factor of activated T cells (NFATc1) in cytoplasm and nucleus were examined by western blot analysis. Interlukin-2 (IL-2) expression in MOLT-4 cells activated by phytohemagglutinin (PHA) was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. IL-2 secretion was measured by an enzyme-linked immunosorbent assay (ELISA). PHA-induced translocation of Rel A and NFATcl to the nucleus were markedly reduced by YGCGT treatment. Furthermore, IL-2 mRNA and protein levels and IL-2 secretion were significantly diminished by YGCGT treatment. In conclusion, YGCGT treatment of T cells inhibits selectively nuclear translocation of RelA and NFATc1, resulting in diminished production of IL-2. These results suggest that YGCGT may have potential as immunosuppressive drugs with improved efficacy and reduced side effects. PMID: 15658604 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: J Immunol. 2005 Jan 15;174(2):983-91. Reductions in I kappa B epsilon and changes in NF-kappa B activity during B lymphocyte differentiation. Doerre S, Mesires KP, Daley KM, McCarty T, Knoetig S, Corley RB. Department of Microbiology, Boston University School of Medicine, Boston, MA 02118, USA. The levels and stability of IkappaBepsilon have been examined in unstimulated and stimulated splenic B cells and compared with that of IkappaBalpha and IkappaBbeta. Primary murine splenic B cells but not T cells were found to contain high levels of IkappaBepsilon protein, equivalent to levels of the abundant IkappaBalpha. Most agents that activate IkappaBalpha and IkappaBbeta degradation do not induce rapid degradation of IkappaBepsilon. Interestingly, however, the levels of IkappaBepsilon, but not of IkappaBalpha or IkappaBbeta, are dramatically reduced upon the stimulation of B cells both in vivo and in vitro. Since IkappaBepsilon exhibits substrate specificity for NF-kappaB Rel homodimers, this suggested the possibility that changes in NF-kappaB-responsive genes might also occur during this transition. Consistent with this hypothesis, we found that a NF-kappaB reporter construct sensitive to p65/RelA homodimers is activated at the time that IkappaBepsilon levels decline following B cell stimulation. In IgG(+) B cell lines, which contain low levels of IkappaBepsilon, this same reporter construct was inactive, suggesting that the increases in Rel homodimer activity that accompany B cell stimulation are transient. However, there are differences in the level of expression of NF-kappaB-responsive genes in these IgG(+) B cell lines compared with their IgM(+) counterparts. From these data, we conclude that there are transient changes in NF-kappaB activity due to reductions in IkappaBepsilon, which might contribute to long-term, persistent changes that accompany B cell differentiation. We propose an important role for IkappaBepsilon in the differential regulation of nuclear NF-kappaB activity in stimulated B cells. PMID: 15634922 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Brain Res Dev Brain Res. 2005 Jan 1;154(1):81-9. Members of the NF-kappaB family expressed in zones of active neurogenesis in the postnatal and adult mouse brain. Denis-Donini S, Caprini A, Frassoni C, Grilli M. Department of Biology, Section of Zoology and Cytology, University of Milan, Milan, Italy. suzanne.donini@unimi.it The Rel/NF-kappaB family of transcription factors is implicated in cell proliferation, cell death, cell migration and cell interactions. Here, we examined by immunohistochemistry the expression pattern of various members of this family during postnatal telencephalon development and during adulthood, and we used neuronal and glial markers to identify the cells types where they are expressed. Distinct Rel/NF-kappaB proteins are highly expressed postnatally in the subventricular zone and in the rostral migratory stream. In particular, Rel A and p50 are expressed in radial glial cells, in migrating neuron precursors and in a population belonging to the astrocytic lineage. Rel B, on the other hand, is only expressed in migrating neuron precursors, whereas c-Rel is present in a few cells located at the edges of the rostral migratory stream. The expression of Rel A and p50 persists into adulthood, particularly in subventricular zone astrocyte-like cells and in migrating neuron precursors, respectively. The selective expression of NF-kappaB members in the postnatal subventricular zone and rostral migratory stream and their persistence into adulthood in regions of ongoing neurogenesis suggests possible mechanisms linking NF-kappaB expression with cell proliferation and migration. Their presence in actively proliferating progenitor cells, detected by BrdU staining, further suggests that NF-kappaB may be part of a signaling pathway that is important for neurogenesis. PMID: 15617758 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Eur J Cancer. 2004 Dec;40(18):2829-36. Rapamycin inhibits doxorubicin-induced NF-kappaB/Rel nuclear activity and enhances the apoptosis of melanoma cells. Romano MF, Avellino R, Petrella A, Bisogni R, Romano S, Venuta S. Department of Biochemistry and Medical Biotechnologies, Federico II University,Via S. Pansini, 5, 80131 Naples, Italy. romano@dbbm.unina.it Inhibition of nuclear factor (NF)-kappaB/Rel can sensitise many tumour cells to death-inducing stimuli, including chemotherapeutic agents, and there are data suggesting that disruption of NF-kappaB may be of therapeutic interest in melanoma. We found that rapamycin sensitised a human melanoma cell line, established from a patient, to the cytolytic effects of doxorubicin. Doxorubicin is a striking NF-kappaB/Rel-inducer, we therefore investigated if rapamycin interfered with the pathway of NF-kappaB/Rel activation, i.e. IkappaBalpha-phosphorylation, -degradation and NF-kappaB/Rel nuclear translocation, and found that the macrolide agent caused a block of IKK kinase activity that was responsible for a reduced nuclear translocation of transcription factors. Western blots for Bcl-2 and c-IAP1 showed increased levels of these anti-apoptotic proteins in cells incubated with doxorubicin, in accordance with NF-kappaB/Rel activation, while rapamycin clearly downmodulated these proteins, in line with its pro-apoptotic ability. The effect of the macrolide on NF-kappa B/Rel induction appeared to be independent of the block in the PI3k/Akt pathway, because it could not be reproduced by the phosphatidyl inositol 3 kinase (PI3k) inhibitor, wortmannin. Recently, the immunophilin, FKBP51, has been shown to be essential for the function of IKK kinase. We found high expression levels of FKBP51 in melanoma cells. Moreover, we confirmed the involvement of this immunophilin in the control of IKK activity. Indeed, IkappaBalpha could not be degraded when FKBP51 was downmodulated by short-interfering RNAs (siRNAs). These findings provide a possible mechanism for the downmodulation of NF-kappaB by rapamycin, since the macrolide agent specifically inhibits FKBP51 isomerase activity. In conclusion, our study demonstrates that rapamycin blocked NF-kappaB/Rel activation independently of PI3k/Akt inhibition suggesting that the macrolide agent could synergise with NF-kappaB-inducing anti-cancer drugs in PTEN-positive tumours. PMID: 15571967 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Clin Endocrinol Metab. 2004 Nov;89(11):5683-93. Expression and deoxyribonucleic acid-binding activity of the nuclear factor kappaB family in the human myometrium during pregnancy and labor. Chapman NR, Europe-Finner GN, Robson SC. School of Surgical and Reproductive Sciences (Obstetrics and Gynaecology), Third Floor, Faculty of Medical Sciences, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, United Kingdom. n.r.chapman@ncl.ac.uk. In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory factors, many of which are regulated by the nuclear factor kappaB (NF-kappaB) family of transcription factors. The expression and DNA-binding activity of NF-kappaB in the myometrium during gestation and parturition were examined. Levels of c-Rel, p50, and p105 NF-kappaB species were dramatically reduced in pregnant myometrium compared with nonpregnant (NP) controls, whereas expression of the RelA subunit remained uniform. Importantly, during labor, expression of all subunits was observed to be significantly reduced in all myometrial samples studied relative to NP levels. Moreover, for RelA, c-Rel, and p50 subunits, there was a gradient of expression between laboring upper (corpus) and lower uterine segment myometrium. No RelB or p52 subunits could be detected. EMSAs identified changes in NF-kappaB subunit composition in the myometrium during pregnancy and labor, with p50 homodimers predominant in NP tissues being replaced with RelA:p50 heterodimers in pregnant and laboring samples. Significantly, RelA was observed to be phosphorylated at serine-536, implicating the involvement of the phosphatidylinositol-3-kinase/AKT pathway in NF-kappaB function in the myometrium. PMID: 15531529 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Brain Res Mol Brain Res. 2004 Nov 24;131(1-2):8-16. Nuclear factor-kappa B p65 in NMDA-induced retinal neurotoxicity. Kitaoka Y, Kumai T, Kitaoka Y, Lam TT, Munemasa Y, Isenoumi K, Motoki M, Kuribayashi K, Kogo J, Kobayashi S, Ueno S. Department of Ophthalmology, St Marianna University School of Medicine, Kawasaki-shi, Kanagawa, Japan. kitaoka@marianna-u.ac.jp Transcription factors of the nuclear factor-kappa B (NF-kappaB)/Rel family may be involved in neuronal cell death or survival. We examined the role of NF-kappaB p65 in N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed that elevated levels of retinal NF-kappaB p65 protein at days 1 and 5 after intravitreal NMDA injection. Immunohistochemistry localized increased NF-kappaB p65 immunoreactivity in the ganglion cell layer (GCL) and the inner nuclear layer (INL) after NMDA injection especially in retinal ganglion cells (RGCs), displaced amacrine cells, and amacrine cells. Concomitant with the early increase in NF-kappaB p65 protein levels, there was an increase in NF-kappaB DNA binding activity after NMDA injection as shown by electrophoretic mobility shift assay (EMSA). These increases in NF-kappaB p65 protein levels and NF-kappaB DNA binding activity were totally abolished by simultaneous injection of NF-kappaB p65 antisense oligodeoxynucleotide (AS ODN). A partial but significant protective effect on the inner retina was noted when the AS ODN was given together with NMDA as shown by morphological analysis, morphometry of cells in the GCL and morphometry of inner plexiform layer thickness as well as quantitative real-time PCR of Thy-1 mRNA levels. These results suggest that activated NF-kappaB p65 may participate in NMDA-induced retinal neuronal cell death and that inhibition of NF-kappaB activation such as the use of AS ODN may be a viable neuroprotective strategy for protective RGCs and other inner retinal neurons. PMID: 15530647 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Cells. 2004 Oct 31;18(2):177-85. Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation. Chang JH, Ryang YS, Morio T, Lee SK, Chang EJ. Department of Biotechnology, College of Engineering and Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea. Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity. PMID: 15528993 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Biol Chem. 2005 Jan 7;280(1):244-52. Epub 2004 Oct 29. cis-acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-kappa B. Anrather J, Racchumi G, Iadecola C. Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021, USA. joa@med.cornell.edu Phosphorylation of nuclear factor-kappa B (NF-kappa B) subunits emerges as a mechanism by which transcriptional activity of nuclear NF-kappa B complexes is regulated in an inhibitor kappa B-independent fashion. As the main transactivator, the p65 subunit of NF-kappa B has an outstanding position in the hierarchy of NF-kappa B proteins. p65 is a multiply phosphorylated protein with phosphorylation sites in the C-terminal transactivation domain and the N-terminal Rel homology domain (RHD). In this study, we describe two previously non-reported phospho-acceptor sites within the p65 RHD. We show that differential phosphorylation of serine residues within the RHD modulates transcriptional activity in a cis-acting element and promoter-specific context, thus leading to a phosphorylation state-dependent gene expression profile. RelA(-/-) mouse embryonic fibroblasts reconstituted with wild-type p65 or p65 phosphorylation-deficient mutants showed a distinctive expression profile of synthetic kappa B-dependent reporters as well as endogenous genes. Hypophosphorylated p65 did not display cis-acting element-specific changes in DNA binding or dimerization behavior. This study shows for the first time that site-specific phosphorylation can target a transcription factor to a particular subset of genes. PMID: 15516339 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Cancer Res. 2004 Oct 15;64(20):7248-55. The nuclear factor kappaB subunits RelA/p65 and c-Rel potentiate but are not required for Ras-induced cellular transformation. Hanson JL, Hawke NA, Kashatus D, Baldwin AS. Lineberger Comprehensive Cancer Center, Curriculum in Genetics and Molecular Biology, and Department of Biology, University of North Carolina, Chapel Hill, North Carolina, USA. Extensive data indicate that oncoproteins, such as oncogenic H-Ras, initiate signal transduction cascades that ultimately lead to the activation of specific transcription factors. We and others have previously demonstrated that Ras activates the inherent transcriptional activation function of the transcription factor nuclear factor kappaB (NF-kappaB). Supportive of the importance of NF-kappaB in transformation, Ras-induced cellular transformation can be suppressed by expression of IkappaBalpha, an inhibitor of NF-kappaB, or by dominant-negative forms of the upstream activator IkappaB kinase (IKK). However, conclusive evidence for a requirement for NF-kappaB subunits in oncogenic transformation has not been reported. Furthermore, there is little understanding of the gene targets controlled by NF-kappaB that might support oncogenic conversion. The data presented here demonstrate that, although both p65 and c-Rel enhance the frequency of Ras-induced cellular transformation, these NF-kappaB subunits are not essential for Ras to transform spontaneously immortalized murine fibroblasts. Microarray analysis identified a set of genes induced by Ras that is dependent on NF-kappaB for their expression and that likely play contributory roles in promoting Ras-induced oncogenic transformation. PMID: 15492243 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Immunity. 2004 Jul;21(1):19-30. The mitogen-induced increase in T cell size involves PKC and NFAT activation of Rel/NF-kappaB-dependent c-myc expression. Grumont R, Lock P, Mollinari M, Shannon FM, Moore A, Gerondakis S. The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. Cell growth during the G1 stage of the cell cycle is partly controlled by inducing c-myc expression, which in B cells is regulated by the NF-kappaB1 and c-Rel transcription factors. Here, we show that c-myc-dependent growth during T cell activation requires c-Rel and RelA and that blocking this growth by inhibiting protein kinase C theta (PKCtheta) coincides with a failure to upregulate c-myc due to impaired RelA nuclear import and inhibition of NFAT-dependent c-rel transcription. These results demonstrate that different Rel/NF-kappaB dimers regulate the mitogenic growth of mature T and B cells, with a signaling pathway incorporating PKCtheta and NFAT controlling c-Rel/RelA-induced c-myc expression in activated T cells. PMID: 15345217 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Neoplasia. 2004 Jul-Aug;6(4):390-400. Nuclear factor-kappaB/p65 (Rel A) is constitutively activated in human prostate adenocarcinoma and correlates with disease progression. Shukla S, MacLennan GT, Fu P, Patel J, Marengo SR, Resnick MI, Gupta S. Department of Urology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA. Aberrant nuclear factor-kappaB (NF-kappaB) activation has been implicated in the pathogenesis of several human malignancies. In this study, we determined whether NF-kappaB is constitutively activated in human prostate adenocarcinoma, and, if so, whether increased NF-kappaB activation and its binding to DNA influence tumor progression. Using tissue samples obtained during transurethral prostatic resection and paraffin-embedded sections of benign and cancer specimens, we determined the nuclear expression of NF-kappaB/p65 and NF-kappaB/p50, cytoplasmic expression of IkappaBalpha, its phosphorylation, and expression of NF-kappaB-regulated genes, specifically Bcl2, cyclin D1, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF). A progressive increase in the expression of NF-kappaB/p65 (but not of p50) was observed in cancer specimens compared to benign tissue, which correlated with increasing levels of IkappaBalpha and its phosphorylation. NF-kappaB DNA-binding activity increased with increasing tumor grade and the binding complex mainly consisted of NF-kappaB/p65-p50 heterodimers. Immunohistochemical analysis showed enhanced nuclear staining for NF-kappaB/p65 in both high-grade (P <.0001) and low-grade (P <.003) cancer specimens, compared to benign tissue. The nuclear levels of NF-kappaB/p65 correlated with concurrent increase in cytosolic levels of IkappaBalpha along with NF-kappaB-dependent expression of Bcl2, cyclin D1, MMP-9, and VEGF. These results demonstrate that NF-kappaB/p65 is constitutively activated in human prostate adenocarcinoma and is related to tumor progression due to transcriptional regulation of NF-kappaB-responsive genes. Copyright 2004 Neoplasia Press, Inc. PMID: 15256061 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Blood. 2005 Jan 15;105(2):689-96. Epub 2004 Jul 13. STAT3 regulates NF-kappaB recruitment to the IL-12p40 promoter in dendritic cells. Hoentjen F, Sartor RB, Ozaki M, Jobin C. Center for Gastrointestinal Biology and Diseases, University of North Carolina at Chapel Hill, USA. Interleukin-10-deficient (IL-10(-/-)) mice develop an IL-12-mediated intestinal inflammation in the absence of endogenous IL-10. The molecular mechanisms of the dysregulated IL-12 responses in IL-10(-/-) mice are poorly understood. In this study, we investigated the role of nuclear factor-kappa B (NF-kappaB) and signal transducers and activators of transcription 3 (STAT3) in lipopolysaccharide (LPS)-induced IL-12p40 gene expression in bone marrow derived-dendritic cells (BMDCs) isolated from wild-type (WT) and IL-10(-/-) mice. We report higher IL-12p40 mRNA accumulation and protein secretion in LPS-stimulated BMDCs isolated from IL-10(-/-) compared with WT mice. LPS-induced NF-kappaB signaling is similar in IL-10(-/-) and WT BMDCs as measured by IkappaBalpha phosphorylation and degradation, RelA phosphorylation and nuclear translocation, and NF-kappaB transcriptional activity, with no down-regulatory effects of exogenous IL-10. Chromatin immunoprecipitation demonstrated enhanced NF-kappaB (cRel, RelA) binding to the IL-12p40 promoter in IL-10(-/-) but not WT BMDCs. Interestingly, LPS induced STAT3 phosphorylation in WT but not IL-10(-/-) BMDCs, a process blocked by IL-10 receptor blocking antibody. Adenoviral gene delivery of a constitutively active STAT3 but not control green fluorescence protein (GFP) virus blocked LPS-induced IL-12p40 gene expression and cRel recruitment to the IL-12p40 promoter. In conclusion, dysregulated LPS-induced IL-12p40 gene expression in IL-10(-/-) mice is due to enhanced NF-kappaB recruitment to the IL-12p40 promoter in the absence of activated STAT3. PMID: 15251981 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Mol Microbiol. 2004 Jul;53(2):587-97. Delayed-relaxed response explained by hyperactivation of RelE. Christensen SK, Gerdes K. Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. Escherichia coli encodes two rel loci, both of which contribute to the control of synthesis of macromolecules during amino acid starvation. The product of relA (ppGpp synthetase I) is responsible for the synthesis of guanosine tetraphosphate, ppGpp, the signal molecule that exerts stringent control of stable RNA synthesis. The second rel locus, relBE, was identified by mutations in relB that confer a so-called 'delayed-relaxed response' characterized by continued RNA synthesis after a lag period of approximately 10 min after the onset of amino acid starvation. We show here that the delayed-relaxed response is a consequence of hyperactivation of RelE. As in wild-type cells, [ppGpp] increased sharply in relB101 relE cells after the onset of starvation, but returned rapidly to the prestarvation level. RelE is a global inhibitor of translation that is neutralized by RelB by direct protein-protein interaction. Lon protease activates RelE during amino acid starvation by degradation of RelB. We found that mutations in relB that conferred the delayed-relaxed phenotype destabilized RelB. Such mutations confer severe RelE-dependent inhibition of translation during amino acid starvation, indicating hyperactivation of RelE. Hyperactivation of RelE during amino acid starvation was shown directly by measurement of RelE-mediated cleavage of tmRNA. The RelE-mediated shutdown of translation terminated amino acid consumption and explains the rapid restoration of the ppGpp level observed in relB mutant cells. Restoration of the prestarvation level of ppGpp, in turn, allows for the resumption of stable RNA synthesis seen during the delayed-relaxed response. PMID: 15228536 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: J Exp Med. 2004 Jul 5;200(1):107-13. Epub 2004 Jun 28. Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor kappaB response. Saccani S, Marazzi I, Beg AA, Natoli G. Institute for Research in Biomedicine, Via Vela 6, 6500 Bellinzona, Switzerland. Transcription factors of the nuclear factor (NF)-kappaB/Rel family translocate into the nucleus upon degradation of the IkappaBs. Postinduction repression of NF-kappaB activity depends on NF-kappaB-regulated resynthesis of IkappaBalpha, which dissociates NF-kappaB from DNA and exports it to the cytosol. We found that after activation, p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding-dependent manner. If proteasome activity is blocked, NF-kappaB is not promptly removed from some target genes in spite of IkappaBalpha resynthesis and sustained transcription occurs. These results indicate that proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination. PMID: 15226358 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Biochem J. 2004 Sep 1;382(Pt 2):393-409. Functions of NF-kappaB1 and NF-kappaB2 in immune cell biology. Beinke S, Ley SC. Division of Immune Cell Biology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. Two members of the NF-kappaB (nuclear factor kappaB)/Rel transcription factor family, NF-kappaB1 and NF-kappaB2, are produced as precursor proteins, NF-kappaB1 p105 and NF-kappaB2 p100 respectively. These are proteolytically processed by the proteasome to produce the mature transcription factors NF-kappaB1 p50 and NF-kappaB2 p52. p105 and p100 are known to function additionally as IkappaBs (inhibitors of NF-kappaB), which retain associated NF-kappaB subunits in the cytoplasm of unstimulated cells. The present review focuses on the latest advances in research on the function of NF-kappaB1 and NF-kappaB2 in immune cells. NF-kappaB2 p100 processing has recently been shown to be stimulated by a subset of NF-kappaB inducers, including lymphotoxin-beta, B-cell activating factor and CD40 ligand, via a novel signalling pathway. This promotes the nuclear translocation of p52-containing NF-kappaB dimers, which regulate peripheral lymphoid organogenesis and B-lymphocyte differentiation. Increased p100 processing also contributes to the malignant phenotype of certain T- and B-cell lymphomas. NF-kappaB1 has a distinct function from NF-kappaB2, and is important in controlling lymphocyte and macrophage function in immune and inflammatory responses. In contrast with p100, p105 is constitutively processed to p50. However, after stimulation with agonists, such as tumour necrosis factor-alpha and lipopolysaccharide, p105 is completely degraded by the proteasome. This releases associated p50, which translocates into the nucleus to modulate target gene expression. p105 degradation also liberates the p105-associated MAP kinase (mitogen-activated protein kinase) kinase kinase TPL-2 (tumour progression locus-2), which can then activate the ERK (extracellular-signal-regulated kinase)/MAP kinase cascade. Thus, in addition to its role in NF-kappaB activation, p105 functions as a regulator of MAP kinase signalling. Publication Types: Review Review, Tutorial PMID: 15214841 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Mol Cell Biol. 2004 Jul;24(13):5733-45. The transcription factors c-rel and RelA control epidermal development and homeostasis in embryonic and adult skin via distinct mechanisms. Gugasyan R, Voss A, Varigos G, Thomas T, Grumont RJ, Kaur P, Grigoriadis G, Gerondakis S. The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. Determining the roles of Rel/NF-kappaB transcription factors in mouse skin development with loss-of-function mutants has been limited by redundancy among these proteins and by embryonic lethality associated with the absence of RelA. Using mice lacking RelA and c-rel, which survive throughout embryogenesis on a tumor necrosis factor alpha (TNF-alpha)-deficient background (rela(-/-) c-rel(-/-) tnfalpha(-/-)), we show that c-rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G(1) progression, and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed that the mutant epidermis develops a TNF-alpha-dependent hyperproliferative condition. Collectively, our findings indicate that RelA and c-rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a postnatal environment through the suppression of innate immune-mediated inflammation. PMID: 15199130 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Arthritis Rheum. 2004 Jun;50(6):1781-7. Increased synovial tissue NF-kappa B1 expression at sites adjacent to the cartilage-pannus junction in rheumatoid arthritis. Benito MJ, Murphy E, Murphy EP, van den Berg WB, FitzGerald O, Bresnihan B. St. Vincent's University Hospital, Elm Park, Dublin, Ireland. OBJECTIVE: To compare the expression of the Rel/NF-kappa B subunits, NF-kappa B1 (p50) and RelA (p65), in paired synovial tissue samples selected from sites adjacent to and remote from the cartilage-pannus junction (CPJ) in patients with inflammatory arthritis. METHODS: Synovial tissue was selected at arthroscopy from sites adjacent to the CPJ and from the suprapatellar pouch of patients who were referred to an early arthritis clinic. Tissue samples from patients with osteoarthritis (OA) undergoing knee arthroplasty were also studied. Rel/NF-kappa B subunit activation and expression were measured by electrophoretic mobility shift assay and supershift analyses and by immunohistochemistry. RESULTS: Tissue samples were obtained from 10 patients with rheumatoid arthritis (RA), 7 with a seronegative arthropathy (SnA), and 6 with OA. Rel/NF-kappa B was abundantly expressed in all samples. In both RA and SnA synovial tissue, the absolute number of NF-kappa B1+ cells at the CPJ was significantly higher than at non-CPJ sites (P = 0.006 and P = 0.02, respectively). The proportion of cells expressing NF-kappa B1 was also significantly higher at the CPJ compared with non-CPJ sites (P = 0.003 in RA, P = 0.009 in SnA). The numbers of RelA+ cells were consistently lower throughout. In RA synovial tissue, but not in SnA synovial tissue, both the absolute number and the proportion of RelA+ cells were significantly higher at the CPJ than at non-CPJ sites (P = 0.003 and P = 0.01, respectively). In OA synovial tissue, the numbers of cells expressing NF-kappa B1 and RelA were similar to those observed at the non-CPJ sites in all inflammatory tissues studied. CONCLUSION: In this study of early inflammatory arthritis, expression of NF-kappa B1 in synovial tissue was highest at sites most likely to be associated with joint erosion. These observations are consistent with a critical role of NF-kappa B1 in joint destruction, and support the rationale for specific therapeutic inhibition of NF-kappa B in RA. PMID: 15188354 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Int J Radiat Biol. 2004 Feb;80(2):115-23. Dose-dependent biphasic induction and transcriptional activity of nuclear factor kappa B (NF-kappaB) in EA.hy.926 endothelial cells after low-dose X-irradiation. Rodel F, Hantschel M, Hildebrandt G, Schultze-Mosgau S, Rodel C, Herrmann M, Sauer R, Voll RE. Department of Radiooncology, University of Erlangen-Nuremberg, Universitatsstrasse 27, D-91054 Erlangen, Germany. franz.rodel@strahlen.imed.uni-erlangen.de PURPOSE: Low-dose radiotherapy is known to exert an anti-inflammatory effect, but the underlying radiobiological mechanisms are still elusive. It was recently reported that transforming growth factor (TGF) beta1 essentially contributes to the reduced adhesion of peripheral blood mononuclear cells to endothelial cells at low-dose X-irradiation. As the transcription factor nuclear factor kappa B (NF-kappaB) is crucially involved in mediating an inflammatory response by inducing the expression of cytokines and adhesion molecules, NF-kappaB DNA binding and transcriptional activity as well as its impact on the expression of TGF-beta1 in EA.hy.926 endothelial cells were analysed subsequently to low-dose radiotherapy. MATERIALS AND METHODS: Human EA.hy.926 endothelial cells were grown to subconfluence. Twenty hours after X-irradiation with single doses ranging from 0.3 to 3 Gy, the cells were activated with tumour necrosis factor-alpha. Four hours later, the cells were harvested. NF-kappaB DNA-binding activity of nuclear extracts was analysed by electrophoretic mobility shift assay. The NF-kappaB subunits p50, p65/RelA, c-Rel and RelB of the NF-kappaB complexes were quantified by enzyme-linked immunoabsorbant assay. The transcriptional activity of NF-kappaB was measured using luciferase reporter gene assays in EA.hy.926 endothelial cells transiently transfected with the plasmid pB2xLuc. To correlate transcriptional activity to TGF-beta1 expression, NF-kappaB decoy oligonucleotides were used to inhibit NF-kappaB activity and TGF-beta1 secretion. RESULTS: After low-dose radiotherapy, an increased NF-kappaB DNA-binding activity was observed in stimulated EA.hy.926 endothelial cells with a relative maximum (threefold induction) at 0.5 Gy. The NF-kappaB activation then decreased after X-irradiation at 0.6-0.8 Gy and subsequently increased again at doses of 1 and 3 Gy. This biphasic induction profile of NF-kappaB was confirmed by the analysis of the NF-kappaB-specific transcriptional activity. The latter showed a relative maximum at 0.5 Gy, a relative minimum between 0.5 and 1.0 Gy, and an increase at 3 Gy. Transfection of EA.hy.926 endothelial cells with NF-kappaB decoy oligonucleotides before irradiation resulted in a 50% reduction of TGF-beta1 secretion at 0.5 Gy compared with control oligonucleotides or untreated cells. CONCLUSIONS: Low-dose radiotherapy induces a biphasic activation of NF-kappaB with a relative maximum at 0.5 Gy. The induction by NF-kappaB of TGF-beta1 in endothelial cells might contribute to the anti-inflammatory properties of low-dose ionizing irradiation. PMID: 15164793 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Zhonghua Er Ke Za Zhi. 2004 Apr;42(4):275-9. [Relativity of nuclear factor-kappaB (P65/Rel-A) and angiotensin II type 1 receptor expression in early stage of lesions of adriamycin nephrosis in young rats and the effects of intervention] [Article in Chinese] Ma H, Li Z, Meng QH, Li XH, Wang XH, Li H, Li WW. Department of Pediatrics, First Hospital, Shanxi Medical University, Taiyuan 030001, China. OBJECTIVE: To investigate the trend and potential pathogenic role of nuclear factor (NF)-kappaB P(65)/Rel-A mRNA and angiotensin-II (AngII) receptor type 1 (AT(1)) proteins expression, and the relativity between them in early stage of renal tubulointerstitial lesions in young rats with adriamycin nephrosis and the interfering effects of treatment with angiotensin converting enzyme inhibitor (ACEI) benazepril and ACEI combined with AngII type 1 receptor antagonist (AT(1)RA) Losartan. METHODS: Male young Wistar rats with adriamycin nephrosis were used as experimental models. At different time points (weeks 1, 2, and 3 in early nephritic phase, the urinary protein and blood biochemical parameters were measured, and P(65)/Rel-A mRNA was detected; AT(1) protein expression was determined by in situ hybridization and immunohistochemical methods. The relativity between them was evaluated. RESULTS: In the early phase of tubulointerstitial lesions, following adriamycin injection and proteinuria aggravated progressively, at week 3, the proteinuria level had reached heavy proteinuria (123.2 +/- 7.7 mg/24 h). The serum parameters reflecting renal function were elevated. The inflammatory cells infiltrated into renal tissues, especially in tubulointerstitial regions, were increased markedly. Swelling of tubular epithelial cells, broadened tubulointerstitial areas, and protein casts in tubule were observed. In situ hybridization and immunochemical staining showed that AT(1) protein was expressed in tubular epithelial cell cytoplasm and on nuclear membranes (AT(1): 1st week 19.8 +/- 1.1%, 2nd week 25.0 +/- 2.6%, 3rd week 37.1 +/- 1.0% (control: 10.3 +/- 0.8%, 10.4 +/- 1.6%, 10.2 +/- 1.5%); and P(65)/Rel-A mRNA expression in the same locations was upregulated. P(65)/Rel-A translocation from cytoplasm into nucleus increased markedly simultaneously. The positive signal of hybridization dominated in cytoplasm gradually became dominant in the nuclei as the pathological changes progressed. The semiquantitative expression of P(65)/Rel-A was 24.0 +/- 3.3% at week 1, 34.2 +/- 2.4% at week 2, 39.9 +/- 6.4% at week 3, while the values of controls were 8.5 +/- 0.4%, 8.7 +/- 1.0%, and 8.4 +/- 0.9%, respectively. There was a positive correlation between AT(1) and P(65)/Rel-A expression in localization and time phase (r = 0.857, P < 0.01). However, the tendency of those factor's expression was all decreased in each treated group, the semiquantitative results were AT(1): 14.6 +/- 2.1%, 13.7 +/- 2.3%, 11.4 +/- 1.1%; P(65)/Rel-A: 18.5 +/- 3.4%, 22.8 +/- 1.6%, 26.7 +/- 4.9% at 1, 2, 3 weeks in ACEI treated group; AT(1): 12.4 +/- 1.5%, 11.1 +/- 1.0%, 10.3 +/- 0.8%; P(65)/Rel-A: 17.9 +/- 5.0%, 21.3 +/- 6.0%, 22.5 +/- 2.5% in AT(1)RA (Losartan) group, respectively. The significant difference were observed between all groups in different time points (P < 0.05). CONCLUSIONS: The present study suggested that NF-kappaB (P(65)/Rel-A) mRNA expression and its activity was enhanced significantly that synchronized with aggravating injures in tubulointerstitial lesions initial period induced by proteinuria-loading in nephrotic young rats. This tendency was related with AngII and its receptors system that may accelerate lesions progressing in many renal diseases. PMID: 15157388 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: J Biol Chem. 2004 Jun 4;279(23):24873-80. Epub 2004 Mar 26. PIAS3 suppresses NF-kappaB-mediated transcription by interacting with the p65/RelA subunit. Jang HD, Yoon K, Shin YJ, Kim J, Lee SY. Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea. Nuclear factor-kappaB (NF-kappaB) is a transcription factor critical for key cellular processes, including immune response, apoptosis, and cell cycle progression. A yeast two-hybrid screening, using the Rel homology domain (RHD) of the p65 subunit (RelA) of NF-kappaB as bait, led to the isolation of PIAS3, previously identified as a specific inhibitor of STAT3. We show that PIAS3 can directly associate with p65 using an in vitro pull-down and in vivo coimmunoprecipitation assays. When overexpressed, PIAS3 inhibits NF-kappaB-dependent transcription induced by treatment with tumor necrosis factor alpha (TNF-alpha) or interleukin-1beta or by overexpression of TNF family receptors such as RANK, TNFR1, and CD30 or signal transducers of TNF receptor-associated factors (TRAFs), including TRAF2, TRAF5, and TRAF6. Downregulation of PIAS3 by RNA interference reverses its effect on TNF-alpha-mediated NF-kappaB activation. We found that an N-terminal region of PIAS3 is necessary for both the interaction with p65 and the transcriptional suppression activity. In addition, we found that an LXXLL coregulator signature motif located within the N-terminal region of PIAS3 is the minimal requirement for the interaction with p65. Furthermore, we demonstrate that PIAS3 interferes with p65 binding to the CBP coactivator, thereby resulting in a decreased NF-kappaB-dependent transcription. Taken together, these data suggest that PIAS3 may function in vivo as a modulator in suppressing the transcriptional activity of p65. PMID: 15140884 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Int Immunopharmacol. 2004 Jun;4(6):763-78. Expression profiles of genes involved in the mouse nuclear factor-kappa B signal transduction pathway are modulated by mangiferin. Leiro J, Arranz JA, Yanez M, Ubeira FM, Sanmartin ML, Orallo F. Laboratorio de Parasitologia, Instituto de Investigacion y Analisis Alimentarios, Universidad de Santiago de Compostela, C/ Constantino Candeira s/n, 15782 Santiago de Compostela, La Coruna, Spain. mpleiro@usc.es The polyphenol mangiferin (MA) has been shown to have various effects on macrophage function, including inhibition of phagocytic activity and of free radical production. To further characterize the immunomodulatory activity of MA, this study investigated its effects on expression by activated mouse macrophages of diverse genes related to the NF-kappaB signaling pathway, using a DNA hybridization array containing 96 NF-kappaB-related genes and on cytokine levels using a cytokine protein array. MA at 10 microM significantly inhibited the expression of (a) two genes of the Rel/NF-kappaB/IkappaB family, RelA and RelB (=I-rel), indicating an inhibitory effect on NF-kappaB-mediated signal transduction; (b) TNF receptor-associated factor 6 (Traf6), indicating probable blockage of activation of the NF-kappaB pathway by lipopolysaccharide (LPS), tumor necrosis factor (TNF), and interleukin 1 (IL-1); (c) other proteins involved in responses to TNF and in apoptotic pathways triggered by DNA damage, including the TNF receptor (TNF-R), the TNF-receptor-associated death domain (TRADD), and the receptor interacting protein (RIP); (d) the extracellular ligand IL-1alpha, again indicating likely interference with responses to IL-1; (e) the pro-inflammatory cytokines IL-1, IL-6, IL-12, TNF-alpha and RANTES (CCL5), and cytokines produced by monocytes and macrophages, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF); (f) other toll-like receptor proteins (in addition to Traf6), including JNK1, JNK2 and Tab1; (g) Scya2 (small inducible cytokine A2=monocyte chemoattractant protein 1); and (h) various intracellular adhesion molecules (ICAMs), and the vascular cell adhesion molecule VCAM-1, which is locally increased in atheromas. The inhibition of JNK1, together with stimulation of c-JUN (i.e. the Jun oncogene) and the previously reported superoxide-scavenging activity of MA, suggests that MA may protect cells against oxidative damage and mutagenesis. Taken together, these results indicate that MA modulates the expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorogenesis, inflammation and various autoimmune diseases, and raise the possibility that it may be of value in the treatment of inflammatory diseases and/or cancer. Copyright 2004 Elsevier B.V. PMID: 15135318 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Clin Cancer Res. 2004 May 1;10(9):3169-78. Suppression of constitutive and tumor necrosis factor alpha-induced nuclear factor (NF)-kappaB activation and induction of apoptosis by apigenin in human prostate carcinoma PC-3 cells: correlation with down-regulation of NF-kappaB-responsive genes. Shukla S, Gupta S. Department of Urology, The James and Eillen Dicke Research Laboratory, Case Western Reserve University, Cleveland, OH 44106, USA. PURPOSE: Development of androgen independence and resistance to apoptosis in prostate cancer are often correlated with high levels of serum tumor necrosis factor (TNF)-alpha in these patients. The loss of sensitivity to TNF-alpha-induced apoptosis in androgen-insensitive prostate carcinoma cells is due in part to constitutive activation of Rel/nuclear factor (NF)-kappaB transcription factors that regulate several cell survival and antiapoptotic genes. Our previous studies have demonstrated growth inhibitory and apoptotic effects of apigenin, a common plant flavonoid, in a variety of human prostate carcinoma cells. Here we examined whether apigenin is effective in inhibiting NF-kappaB expression in androgen-insensitive human prostate carcinoma cells exhibiting high constitutive levels of NF-kappaB. EXPERIMENTAL DESIGN: Using androgen-insensitive human prostate carcinoma PC-3 cells, the effect of apigenin was assessed on NF-kappaB activation by electrophoretic mobility shift assay and reporter gene assay. Expression of NF-kappaB subunits p65 and p50, IkappaBalpha, p-IkappaBalpha, in-beads kinase assay and NF-kappaB-regulated genes were determined by Western blot analysis. Apoptosis was determined by annexin V/propidium iodide staining after fluorescence-activated cell-sorting analysis. RESULTS: Treatment of cells with 10-40- micro M doses of apigenin inhibited DNA binding and reduced nuclear levels of the p65 and p50 subunits of NF-kappaB. Apigenin inhibited IkappaBalpha degradation and IkappaBalpha phosphorylation and significantly decreased IKKalpha kinase activity. Apigenin also inhibited TNF-alpha-induced activation of NF-kappaB via the IkappaBalpha pathway, thereby sensitizing the cells to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB activation correlated with a decreased expression of NF-kappaB-dependent reporter gene and suppressed expression of NF-kappaB-regulated genes [specifically, Bcl2, cyclin D1, cyclooxygenase-2, matrix metalloproteinase 9, nitric oxide synthase-2 (NOS-2), and vascular endothelial growth factor]. CONCLUSIONS: Our results indicate that inhibition of NF-kappaB by apigenin may lead to prostate cancer suppression by transcriptional repression of NF-kappaB-responsive genes as well as selective sensitization of prostate carcinoma cells to TNF-alpha-induced apoptosis. PMID: 15131058 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: J Surg Res. 2004 May 1;118(1):9-14. Increased expression of NF-kappa B subunits in human pancreatic cancer cells. Chandler NM, Canete JJ, Callery MP. Department of Surgery, University of Massachusetts Medical School, Worcester, MA, USA. mcallery@caregroup.harvard.edu BACKGROUND: Activation of NF-kappa B-dependent antiapoptotic genes may factor in the chemoresistance of pancreatic cancer. It is not known whether NF-kappa B subunit composition changes during oncogenesis and regulates overall NF-kappa B activation. We compared the relative expression of NF-kappa B subunits with nuclear activation of p65 between variably differentiated pancreatic cancer cells. MATERIALS AND METHODS: Proliferating human pancreatic cancer cells (PANC-1, BxPC-3) and nonmalignant intestinal cells (FHS 74 Int) were harvested. Baseline expression of NF-kappa B subunits (p65, p52, p50, c-Rel) and its inhibitor I kappa B-alpha were determined by Western blot. Nuclear NF-kappa B p65 activity was measured by ELISA. Results were analyzed by ANOVA (P < 0.05) and Tukey's HSD for pairwise comparisons when appropriate (P < 0.05). RESULTS: Constitutive expression of NF-kappa B subunits was detected in proliferating, intestinal cells (FHS 74 Int). Both cytoplasmic (I kappa B-alpha, p50, p52, p65) and nuclear (p50, p52, p65, c-Rel) NF-kappa B subunits were significantly increased in both PANC-1 and BxPC-3 cells compared to FHS 74 Int. While nuclear p65 subunit levels were similarly elevated, actual p65 activity was only significantly greater in PANC-1 cells compared to either BxPC-3 or FHS 74 Int (P < 0.05). CONCLUSIONS: Compared to nonmalignant proliferating intestinal cells, these pancreatic cancer cell lines have increased levels of NF-kappa B subunits. Actual nuclear NF-kappa B activity, however, appears to correlate more with degree of tumor differentiation than with NF-kappa B subunit expression. PMID: 15093710 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6098-103. Epub 2004 Apr 12. CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters. Marinari B, Costanzo A, Marzano V, Piccolella E, Tuosto L. Department of Cellular and Developmental Biology, University of Rome La Sapienza, 00185 Rome, Italy. CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IkappaB kinase and NF-kappaB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8 and B cell activation factor, but not proliferative (IL-2), genes, in a NF-kappaB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-kappaB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-kappaB sites on the IL-2 gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation. PMID: 15079071 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Cell. 2004 Apr 2;117(1):57-68. Erratum in: Cell. 2004 Apr 30;117(3):415. Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]. Hogg T, Mechold U, Malke H, Cashel M, Hilgenfeld R. Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis, Rel(Seq), reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2':3'-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites. PMID: 15066282 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Proc Natl Acad Sci U S A. 2004 Apr 13;101(15):5634-9. Epub 2004 Apr 2. Canonical pathway of nuclear factor kappa B activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis. Monaco C, Andreakos E, Kiriakidis S, Mauri C, Bicknell C, Foxwell B, Cheshire N, Paleolog E, Feldmann M. Kennedy Institute of Rheumatology, Imperial College, Charing Cross Campus, London W6 8LH, United Kingdom. Nuclear factor kappa B (NF-kappa B) activation has been observed in human atherosclerotic plaques and is enhanced in unstable coronary plaques, but whether such activation has a protective or pathophysiological role remains to be determined. We addressed this question by developing a short-term culture system of cells isolated from human atherosclerotic tissue, allowing efficient gene transfer to directly investigate signaling pathways in human atherosclerosis. We found that NF-kappa B is activated in these cells and that this activity involves p65, p50, and c-Rel but not p52 or RelB. This NF-kappa B activation can be blocked by overexpression of I kappa B alpha or dominant-negative I kappa B kinase (IKK)-2 but not dominant-negative IKK-1 or NF-kappa B-inducing kinase, resulting in selective inhibition of inflammatory cytokines (tumor necrosis factor alpha, IL-6, and IL-8), tissue factor, and matrix metalloproteinases without affecting the antiinflammatory cytokine IL-10 or tissue inhibitor of matrix metalloproteinases. Our results demonstrate that the canonical pathway of NF-kappa B activation that involves p65, p50, c-Rel, and IKK-2 is activated in human atherosclerosis and results in selective up-regulation of major proinflammatory and prothrombotic mediators of the disease. PMID: 15064395 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Antiviral Res. 2004 Apr;62(1):27-36. A quantitative bioassay for HIV-1 gene expression based on UV activation: effect of glycyrrhizic acid. Cherng JM, Lin HJ, Hsu YH, Hung MS, Lin JC. Department of Internal Medicine, Tzu Chi General Hospital, Hualien, Taiwan, ROC. Previous reports have shown that HIV-LTRcat constructs stably transfected in HeLa cells are inducible after exposure to UV light. We have optimized this system for studying the effect of drugs on HIV-1 gene expression. The maximum UV response was observed in quiescent stationary cells stimulated with fresh medium for 3h. Glycyrrhizic acid suppressed UV-induced HIV gene expression in a concentration-dependent manner. The inhibitory effect was strongest when GL was added immediately after UV exposure; it was still evident when GL was added at 5 h, it was completely lost at 10 h, after UV exposure. The inhibitory effect was even more pronounced if the cells were pretreated with sub-effective dose (0.0012 mM) of GL prior to UV exposure. The IC50 values with and without pretreatment were 0.04 and 0.38 mM, respectively. Cell proliferation and viability were not affected by GL at doses as high as 2.4 mM. The inhibitory effect of GL on UV-induced CAT activity correlated with the complete inhibition of binding activities of NF-kappaB p65, NF-kappaB p50, c-Fos, and c-Rel. Thus, the UV-based bioassay as proposed here can be exploited for the routine screening of the compounds that interfere with HIV-1 gene expression. PMID: 15026199 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: FEBS Lett. 2004 Mar 12;561(1-3):217-22. Novel sites in the p65 subunit of NF-kappaB interact with TFIIB to facilitate NF-kappaB induced transcription. Xia C, Watton S, Nagl S, Samuel J, Lovegrove J, Cheshire J, Woo P. Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, UK. Nuclear factor kappaB (NF-kappaB) transcription factors regulate a large number of genes in response to inflammation, infection and stressful conditions. In this study, we investigated whether NF-kappaB p65 regulates the transcription of target genes by interacting with components of the basal transcription machinery. We examined the interaction of p65 with the basal transcription factor IIB (TFIIB). Glutathione S-transferase pull down assays showed that the Rel homology domain of p65 is important for binding to TFIIB. Molecular modelling, together with the generation of specific point mutants, revealed that residues 41 R and 42 S in the Rel homology domain of p65 facilitate the interaction with TFIIB. Mutation of these residues showed a decrease in p65 induced transcription, suggesting that they are involved in a functional interaction with TFIIB. PMID: 15013781 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: J Biol Chem. 2004 Apr 30;279(18):18137-45. Epub 2004 Feb 12. Human T-cell lymphotropic virus type 1 tax induction of biologically Active NF-kappaB requires IkappaB kinase-1-mediated phosphorylation of RelA/p65. O'Mahony AM, Montano M, Van Beneden K, Chen LF, Greene WC. Gladstone Institute for Virology and Immunology, Department of Medicine, University of California, San Francisco, California 94141, USA. Activation of the NF-kappaB/Rel family of transcription factors proceeds through a catalytic complex containing IkappaB kinase (IKK)-1 and IKK2. Targeted disruption of each of the IKK genes suggests that these two kinases may mediate distinct functions in the activation pathway. In our studies of the human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein, we have uncovered a new function of IKK1 required for complete activation of the NF-kappaB transcriptional program. In IKK1(-/-) murine embryonic fibroblasts (MEFs), Tax normally induced early NF-kappaB activation events. However, NF-kappaB induced by Tax in these IKK1(-/-) cells was functionally impaired. In IKK1(-/-) (but not wild-type) MEFs, Tax failed to activate several different kappaB reporter constructs or to induce the endogenous IkappaBalpha gene. In contrast, Tax normally activated the cAMP-responsive element-binding protein/activating transcription factor pathway, leading to full stimulation of an HTLV-1 long terminal repeat reporter construct in IKK1(-/-) cells. Furthermore, reconstitution of IKK1(-/-) cells with kinase-proficient (but not kinase-deficient) forms of IKK1 restored the Tax induction of full NF-kappaB transactivation. We further found that the defect in NF-kappaB action in IKK1(-/-) cells correlated with a failure of Tax to induce phosphorylation of the RelA/p65 subunit of NF-kappaB at Ser(529) and Ser(536). Such phosphorylation of RelA/p65 was readily detected in wild-type MEFs. Phosphorylation of Ser(536) was required for a complete response to Tax expression, whereas phosphorylation of Ser(529) appeared to be less critical. Together, these findings highlight distinct roles for the IKK1 and IKK2 kinases in the activation of NF-kappaB in response to HTLV-1 Tax. IKK2 plays a dominant role in signaling for IkappaBalpha degradation, whereas IKK1 appears to play an important role in enhancing the transcriptional activity of NF-kappaB by promoting RelA/p65 phosphorylation. PMID: 14963024 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: J Biol Chem. 2004 Apr 23;279(17):17819-25. Epub 2004 Feb 9. Identification of a ZU5 and death domain-containing inhibitor of NF-kappaB. Zhang J, Xu LG, Han KJ, Shu HB. Department of Immunology, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, Colorado 80206, USA. The transcription factor NF-kappaB plays important roles in inflammation and cell survival. NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB family members, including p65 (RelA), c-Rel (Rel), RelB, NF-kappaB1/p50, and NF-kappaB2/p52. Here we report the identification and characterization of a novel ZU5 and death domain-containing protein designated ZUD. In reporter gene assays, overexpression of ZUD inhibited NF-kappaB-dependent transcription induced by both tumor necrosis factor (TNF) and interleukin-1 and their downstream signaling proteins. Gel shift assays indicated that the overexpression of ZUD inhibited binding of NF-kappaB to its target sequence. ZUD is a cytoplasmic protein, and coimmunoprecipitation assays indicated that ZUD interacted with the NF-kappaB subunit p105 and transactivator p65. Consistent with its role in inhibition of NF-kappaB-dependent transcription, ZUD sensitized cells to apoptosis induced by TNF and the TNF-related apoptosis-inducing ligand (TRAIL). Our findings suggest that ZUD is an inhibitor of NF-kappaB activation and that this protein may provide an alternative regulatory mechanism for NF-kappaB-mediated transcription. PMID: 14769797 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Biol Chem. 2004 Mar 26;279(13):12819-26. Epub 2004 Jan 1. A p105-based inhibitor broadly represses NF-kappa B activities. Fu D, Kobayashi M, Lin L. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. An IkappaBalpha-based NF-kappaB super repressor (sr) has been used widely for studying genes regulated by NF-kappaB transcription factors. Repression of NF-kappaB by IkappaBalpha(sr) also facilitates tumor necrosis factor alpha-induced apoptosis in the cell. However, IkappaBalpha primarily targets RelA and c-Rel-containing complexes, leaving other NF-kappaB/Rel protein complexes, such as p50 and p52 homodimers, and RelB heterodimers uninhibited. Because these atypical NF-kappaB complexes also contribute to gene regulation and are activated in pathological conditions, broad inhibition of all NF-kappaB species is of significant pharmacological and clinical interests. We have designed, generated, and tested a p105-based NF-kappaB super repressor. We showed that p105(sr), which no longer generates p50 and undergoes signal-induced degradation, effectively inhibits all NF-kappaB activities. In addition, we also demonstrated that p105(sr) significantly enhances tumor necrosis factor alpha-mediated killing of MT1/2 skin papilloma cells where p50 homodimer activity is elevated. Our results suggest that p105(sr) is a broader range and effective NF-kappaB super repressor and can potentially be used in cells where a noncanonical NF-kappaB activity is dominant or multiple NF-kappaB activities are activated. PMID: 14703515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Environ Health. 2003 Dec 16;2(1):16. Aryl hydrocarbon receptor (AhR) agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study. Jensen BA, Leeman RJ, Schlezinger JJ, Sherr DH. Department of Environmental Health, Boston University School of Public Health, 715 Albany Street, Boston, MA 02118, USA. bjensEn@bu.edu BACKGROUND: Bone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses. METHODS: Bone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS) to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA) and quantified by real-time PCR. Cytokine (IL-6) protein production was measured by ELISA. NF-kappaB EMSAs were used to study IL-6 transcriptional regulation. RESULTS: RPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-kappaB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in the LPS-mediated induction of DNA-binding RelA/p50 and c-Rel/p50 heterodimers in the presence of DMBA. CONCLUSIONS: Common environmental AhR agonists can suppress the response to bacterial lipopolysaccharide, a model for innate inflammatory responses, through down-regulation of IL-6, a cytokine critical to the growth of several hematopoietic cell subsets, including early B cells. This suppression occurs at least at the level of IL-6 gene transcription and may be regulated by NF-kappaB. PMID: 14678569 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: J Bone Miner Res. 2003 Dec;18(12):2159-68. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-kappaB activation. Wang C, Steer JH, Joyce DA, Yip KH, Zheng MH, Xu J. Department of Orthopaedics, University of Western Australia, Nedlands, Western Australia, Australia. The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation. INTRODUCTION: The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. MATERIALS AND METHODS: RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. RESULTS: We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. CONCLUSION: Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption. PMID: 14672351 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Oncogene. 2004 Feb 5;23(5):1030-42. Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes. Fan Y, Rayet B, Gelinas C. Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854-5638, USA. rel/nf-kappaB genes are amplified, overexpressed, or constitutively activated in many human hematopoietic tumors; however, the molecular mechanisms by which they contribute to tumorigenesis remain to be determined. Here, we explored the oncogenic potential of cellular Rel/NF-kappaB proteins in vitro and in vivo. We show that overexpression of wild-type mouse and human c-rel genes suffices to malignantly transform primary spleen cells in stringent soft agar assays and produce fatal tumors in vivo. In contrast relA and a constitutively active form of IKKbeta did not. Importantly, a hybrid RelA protein with its C-terminal transactivation domain substituted by that of v-Rel was potently oncogenic in vitro and in vivo. The transactivation domain of v-Rel selectively conferred an oncogenic phenotype upon the Rel homology domain (RHD) of RelA, but not to the more divergent RHDs of p50/NF-kappaB1, p52/NF-kappaB2, or RelB. Collectively, our results highlight important differences in the intrinsic oncogenic activity of mammalian c-Rel and RelA proteins, and indicate that critical determinants of their differential oncogenicity reside in their divergent transactivation domains. These findings provide experimental evidence for a role of mammalian Rel/NF-kappaB factors in leukemia/lymphomagenesis in an in vivo animal model, and are consistent with the implication of c-rel in many human lymphomas. PMID: 14647412 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12301-6. Epub 2003 Oct 6. Inhibition of NF-kappaB by ZAS3, a zinc-finger protein that also binds to the kappaB motif. Hong JW, Allen CE, Wu LC. Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA. The ZAS proteins are large zinc-finger transcriptional proteins implicated in growth, signal transduction, and lymphoid development. Recombinant ZAS fusion proteins containing one of the two DNA-binding domains have been shown to bind specifically to the kappaB motif, but the endogenous ZAS proteins or their physiological functions are largely unknown. The kappaB motif, GGGACTTTCC, is a gene regulatory element found in promoters and enhancers of genes involved in immunity, inflammation, and growth. The Rel family of NF-kappaB, predominantly p65.p50 and p50.p50, are transcription factors well known for inducing gene expression by means of interaction with the kappaB motif during acute-phase responses. A functional link between ZAS and NF-kappaB, two distinct families of kappaB-binding proteins, stems from our previous in vitro studies that show that a representative member, ZAS3, associates with TRAF2, an adaptor molecule in tumor necrosis factor signaling, to inhibit NF-kappaB activation. Biochemical and genetic evidence presented herein shows that ZAS3 encodes major kappaB-binding proteins in B lymphocytes, and that NF-kappaB is constitutively activated in ZAS3-deficient B cells. The data suggest that ZAS3 plays crucial functions in maintaining cellular homeostasis, at least in part by inhibiting NF-kappaB by means of three mechanisms: inhibition of nuclear translocation of p65, competition for kappaB gene regulatory elements, and repression of target gene transcription. PMID: 14530385 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Exp Mol Med. 2003 Aug 31;35(4):290-300. Cytochrome C-dependent Fas-independent apoptotic pathway in HeLa cells induced by delta12-prostaglandin J2. Kim BE, Roh SR, Kim JW, Jeong SW, Kim IK. Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea. ikkim@catholic.ac.kr Cyclopentenone prostaglandins (PGs) have antiproliferative activity on various tumor cell growth in vitro. Particularly, 9-deoxy-delta(9,12)-13,14-dihydro PGD(2) (delta(12)-PGJ(2)) was reported for its antineoplastic and apoptotic effects on various cancer cells, but its mechanism inducing apoptosis is still not clear. In this study, we have characterized apoptosis induced by delta(12)-PGJ(2) in HeLa cells. Treatment of delta(12)-PGJ(2) induced apoptosis as indicated by DNA fragmentation, chromatin condensation, and formation of apoptotic body. We also observed release of cytochrome c from mitochondria and activation of caspase cascade including caspase-3, -8, and -9. And the pan-caspase inhibitor z-Val-Ala-Asp (OMe) fluoromethyl-ketone (z-VAD-fmk) and Q-Val-Asp (OMe)-CH(2)-OPH (Q-VD (OMe)-OPH) prevented cell death induced by delta(12)-PGJ(2) showing participation of caspases in this process. However, protein expression level of Bcl-2 family was not altered by delta(12)-PGJ(2), seems to have no effect on HeLa cell apoptosis. And ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase 8 indicating that Fas receptor-ligand interaction was not involved in this pathway. Treatment of delta(12)-PGJ(2) also leads to suppression of nuclear factor kappaB (NF-kappaB) as indicated by nuclear translocation of p65/RelA and c-Rel and its DNA binding ability analyzed by EMSA. Taken together, our results suggest that delta(12)-PGJ(2)-induced apoptosis in HeLa cell utilized caspase cascade without Fas receptor-ligand interaction and accompanied with NF-kappaB inactivation. PMID: 14508070 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: Immunol Rev. 2003 Oct;195:91-105. Regulation of secondary lymphoid organ development by the nuclear factor-kappaB signal transduction pathway. Weih F, Caamano J. Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, Karlsruhe, Germany. falk.weih@itg.fzk.de In primary lymphoid organs, such as thymus and bone marrow, B and T lymphocytes differentiate from lymphoid stem cells into mature albeit naive effector cells. In contrast, secondary lymphoid organs, such as the spleen, lymph nodes, and Peyer's patches (PPs), provide an environment that enable lymphocytes to interact with each other, with accessory cells, and with antigens, resulting in the initiation of antigen-specific primary immune responses. Recently, the analysis of gene-knockout mice has shed light on the signaling pathways, cellular requirements, and molecular mechanisms involved in secondary lymphoid organ development. In particular, signals that converge on the nuclear factor-kappaB (NF-kappaB) pathway have been demonstrated to play an important role in both early developmental steps as well as maintenance of secondary lymphoid organ structures. Analysis of the histopathological changes in secondary lymphoid tissues of mice lacking individual Rel/NF-kappaB family members, upstream kinases, and receptors strongly indicates that activation of the recently described alternative NF-kappaB pathway by membrane-bound lymphotoxin, via p52-RelB heterodimers, plays a major role during initiation steps of secondary lymphoid organ development. Induction of the classical p50-RelA NF-kappaB activity, as exemplified by tumor necrosis factor receptor signaling, clearly also contributes, but seems to be involved primarily in later developmental step, such as the proper cellular and structural organization of B-cell follicles. Publication Types: Review PMID: 12969313 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: J Biol Chem. 2003 Nov 14;278(46):45994-8. Epub 2003 Aug 28. Phosphorylation of serine 337 of NF-kappaB p50 is critical for DNA binding. Hou S, Guan H, Ricciardi RP. Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. It has been demonstrated that phosphorylation of the p50 subunit of NF-kappaB is required for efficient DNA binding, yet the specific phospho-residues of p50 have not been determined. In this study, we substituted all of the serine and conserved threonine residues in the p50 Rel homology domain and identified three serine residues, Ser65, Ser337, and Ser342, as critical for DNA binding without affecting dimerization. Although substitution with negatively charged aspartic acid at each of these positions failed to restore DNA binding, substitution with threonine, a potential phospho-acceptor, retained DNA binding for residues 65 and 337. In particular, Ser337, in a consensus site for protein kinase A (PKA) and other kinases, was shown to be phosphorylated both in vitro and in vivo. Importantly, phosphorylation of Ser337 by PKA in vitro dramatically increased DNA binding of p50. This study shows for the first time that the DNA binding ability of NF-kappaB p50 subunit is regulated through phosphorylation of residue Ser337, which has implications for both positive and negative control of NF-kappaB transcription. PMID: 12947093 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: J Immunol. 2003 Aug 15;171(4):1816-24. T cell-intrinsic requirement for NF-kappa B induction in postdifferentiation IFN-gamma production and clonal expansion in a Th1 response. Corn RA, Aronica MA, Zhang F, Tong Y, Stanley SA, Kim SR, Stephenson L, Enerson B, McCarthy S, Mora A, Boothby M. Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232, USA. NF-kappaB/Rel transcription factors are linked to innate immune responses and APC activation. Whether and how the induction of NF-kappaB signaling in normal CD4(+) T cells regulates effector function are not well-understood. The liberation of NF-kappaB dimers from inhibitors of kappaB (IkappaBs) constitutes a central checkpoint for physiologic regulation of most forms of NF-kappaB. To investigate the role of NF-kappaB induction in effector T cell responses, we targeted inhibition of the NF-kappaB/Rel pathway specifically to T cells. The Th1 response in vivo is dramatically weakened when T cells defective in their NF-kappaB induction (referred to as IkappaBalpha(DeltaN) transgenic cells) are activated by a normal APC population. Analyses in vivo, and IL-12-supplemented T cell cultures in vitro, reveal that the mechanism underlying this T cell-intrinsic requirement for NF-kappaB involves activation of the IFN-gamma gene in addition to clonal expansion efficiency. The role of NF-kappaB in IFN-gamma gene expression includes a modest decrease in Stat4 activation, T box expressed in T cell levels, and differentiation efficiency along with a more prominent postdifferentiation step. Further, induced expression of Bcl-3, a trans-activating IkappaB-like protein, is decreased in T cells as a consequence of NF-kappaB inhibition. Together, these findings indicate that NF-kappaB induction in T cells regulates efficient clonal expansion, Th1 differentiation, and IFN-gamma production by Th1 lymphocytes at a control point downstream from differentiation. PMID: 12902482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: Mol Cell Biol. 2003 Aug;23(16):5738-54. Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors. Romieu-Mourez R, Kim DW, Shin SM, Demicco EG, Landesman-Bollag E, Seldin DC, Cardiff RD, Sonenshein GE. Department of Biochemistry, Boston University Medical School, Boston, Massachusetts 02118, USA. Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-kappaB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-kappaB. Analysis of the composition of NF-kappaB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-kappaB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-kappaB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis. PMID: 12897145 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: J Biol Chem. 2003 Oct 3;278(40):39242-50. Epub 2003 Jul 21. Critical role of RelB serine 368 for dimerization and p100 stabilization. Maier HJ, Marienfeld R, Wirth T, Baumann B. Department of Physiological Chemistry, Ulm University, Ulm 89081, Germany. In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner. PMID: 12874295 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: Oncogene. 2003 Jul 10;22(28):4356-69. Mechanism of cytosine arabinoside-mediated apoptosis: role of Rel A (p65) dephosphorylation. Sreenivasan Y, Sarkar A, Manna SK. Laboratory of Immunology, Centre for DNA Fingerprinting & Diagnostics, Nacharam, Hyderabad 500 076, India. Nuclear transcription factor kappa B (NF-kappaB) has been shown both to block apoptosis and to promote cell proliferation, and hence has been considered an important target for anticancer drug development. The pyrimidine analogue cytosine arabinoside (araC) is among the most effective agents used in the treatment of acute leukemia, and we demonstrate in this study that its chemotherapeutic activity may be mediated by its inhibition of NF-kappaB. We found that in Jurkat cells, although tumor necrosis factor (TNF), araC, or ceramide induced NF-kappaB, the induction was only transient in the case of araC. In both HuT-78 and serum-activated LPS-stimulated Jurkat (SA-LPS/Jkt) cells that expressed NF-kappaB, TNF or ceramide treatments did not affect the NF-kappaB expression whereas araC downregulated it. AraC, but not TNF or ceramide was able to induce apoptosis in these cells as detected by assays for lipid peroxidation, reactive oxygen intermediates generation, caspase activation, cytotoxicity, Bcl-2 degradation, and DNA fragmentation. AraC also potentiated apoptosis mediated by cis-platin, etoposide, or taxol in these cells. AraC was able to induce protein phosphatases (PP) 2A and 2B-A, and phosphorylation of p65 subunit of NF-kappaB in the HuT-78 and SA-LPS/Jkt cells was downregulated by araC treatment. Furthermore, calyculin A, a specific phospho-serine/phospho-threonine phosphatase inhibitor, protected HuT-78 and SA-LPS/Jkt cells from araC-mediated NF-kappaB downregulation and apoptosis. These observations collectively suggest that araC induces apoptosis in NF-kappaB-expressing cells by dephosphorylating the p65 subunit of NF-kappaB. PMID: 12853972 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Exp Hematol. 2003 Jun;31(6):504-11. Mechanisms of Bcr-Abl-mediated NF-kappaB/Rel activation. Kirchner D, Duyster J, Ottmann O, Schmid RM, Bergmann L, Munzert G. Abteilung Innere Medizin III, Universitat Ulm, Ulm, Germany. Bcr-Abl constitutes a deregulated tyrosine kinase involved in the pathogenesis of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Although activation of the transcription factor NF-kappaB/Rel has been demonstrated, mechanisms of NF-kappaB/Rel activation by Bcr-Abl remain obscure. In this paper we demonstrate activation of NF-kappaB/Rel by Bcr-Abl and for the first time by v-Abl. Furthermore, we investigated mechanisms of NF-kappaB/Rel induction by Bcr-Abl and v-Abl. Both Bcr-Abl and v-Abl induced NF-kappaB/Rel DNA binding in Ba/F3 cells. DNA binding was a result of nuclear translocation of p65/RelA, whereas p65/RelA expression was unaffected. Nuclear translocation of p65/RelA is at least partially due to increased IkappaBalpha degradation, which is independent of IkappaB kinase (IKK) activity. IKK activity is not deregulated by Bcr-Abl and v-Abl. NF-kappaB/Rel transactivation was dependent on abl kinase activity but independent of Grb2 and Grb10 binding tobcr sequences. In addition, NF-kappaB/Rel activation was dependent on Ras activity. Primary CML blasts showed constitutive p65/RelA NF-kappaB/Rel DNA binding activity. Thus NF-kappaB/Rel represents a potential target for molecular therapies in CML. PMID: 12829026 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Mol Cell. 2003 Jun;11(6):1563-74. Modulation of NF-kappaB activity by exchange of dimers. Saccani S, Pantano S, Natoli G. Institute for Research in Biomedicine, Via Vela 6, CH6500 Bellinzona, Switzerland. Transcription factors within a family usually share the ability to recognize similar or identical consensus sites. For example, the five mammalian NF-kappaB/Rel proteins generate more than 12 dimers recognizing 9-11 nucleotide kappaB sites. Each dimer selectively regulates a few target promoters; however, several genes are redundantly induced by more than one dimer. Whether this property simply generates redundancy in target gene activation or underlies more complex regulatory mechanisms is an open issue. We show here that during dendritic cell maturation, rapidly activated dimers (e.g., p50/RelA) bound to a subset of target promoters are gradually replaced by slowly activated dimers (e.g., p52/RelB). Since the dimers have different transcriptional activity at each promoter, the dimer exchange allows fine tuning of the response over time. Further, due to the insensitivity of p52/RelB to the NF-kappaB inhibitors, the IkappaBs, dimer exchange contributes to sustained activation of selected NF-kappaB targets in spite of the resynthesis of IkappaBalpha. PMID: 12820969 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: Int J Cancer. 2003 Jul 20;105(6):735-46. Mitogenic and antiapoptotic role of constitutive NF-kappaB/Rel activity in pancreatic cancer. Liptay S, Weber CK, Ludwig L, Wagner M, Adler G, Schmid RM. Department of Pediatrics, University of Ulm, Ulm, Germany. The transcription factor NF-kappaB/Rel was found to be constitutively activated in human pancreatic cancer. RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF-kappaB/Rel-binding activity consists of NF-kappaB1(p50) and RelA(p65). Constitutive NF-kappaB/Rel activity correlates with IkappaB kinase (IKK) activity and can be blocked by dominant negative mutants of IKKbeta and to a lesser extent by IKKalpha. Constitutive NF-kappaB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3 kinase inhibitor LY294002, or dominant negative mutant Akt kinase. Transfection of a dominant negative mutant epidermal growth factor receptor (EGF-R), EGF-R kinase inhibitor Tyrphostin and LY 294002 blocked IKK activity and NF-kappaB-dependent transcription. Inhibition of constitutive IKK or NF-kappaB/Rel activity increased the number of apoptotic cells. Stably expressing a nondegradable form of IkappaBalpha inhibited anchorage-dependent and -independent proliferation in MiaPaCa2 and Panc1 cells. Our data demonstrate that an EGF-R/Ras/PI3 kinase/Akt/IKK-dependent pathway contributes to constitutive NF-kappaB/Rel activity in pancreatic cancer. Inhibition of NF-kappaB/Rel activity reveals a mitogenic and antiapoptotic role for NF-kappaB/Rel in pancreatic cancer. Copyright 2003 Wiley-Liss, Inc. PMID: 12767057 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: J Biol Chem. 2003 Jul 25;278(30):28181-92. Epub 2003 May 5. Troglitazone antagonizes tumor necrosis factor-alpha-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kappaB. Ruan H, Pownall HJ, Lodish HF. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. Troglitazone (TGZ), a member of the thiazolidinedione class of anti-diabetic compounds and a peroxisome proliferator activator receptor-gamma (PPAR-gamma) agonist, restores systemic insulin sensitivity and improves the full insulin resistance syndrome in vivo. The mechanisms underlying its in vivo function are not understood. Here we investigated the potential functional interaction between PPAR-gamma and NF-kappaB in adipocytes. We show that TGZ selectively blocked tumor necrosis factor-alpha-induced and NF-kappaB-dependent repression of multiple adipocyte-specific genes and induction of growth phase and other genes. This occurs without interfering with NF-kappaB expression, activation, nuclear translocation, or DNA binding and without suppressing NF-kappaB-dependent survival signals. Notably, the expressions of some tumor necrosis factor-alpha-induced genes in adipocytes were unaffected by PPAR-gamma activation. In reporter gene assays in HeLa cells, ectopic expression of PPAR-gamma abolished induction of a NF-kappaB-responsive reporter gene by the p65 subunit (RelA) of NF-kappaB, and the inhibition was further enhanced in the presence of TGZ. Conversely, overexpression of p65 inhibited induction of a PPAR-gamma-responsive reporter gene by activated PPAR-gamma in a dose-dependent manner. The inhibitory effect was independent of the presence of NF-kappaB-binding sites in the promoter region. Other NF-kappaB family members, p50 and c-Rel as well as the S276A mutant of p65, blocked PPAR-gamma-mediated gene transcription less effectively. Thus, p65 antagonizes the transcriptional regulatory activity of PPAR-gamma in adipocytes, and PPAR-gamma activation can at least partially override the inhibitory effects of p65 on the expression of key adipocyte genes. Our data suggest that inhibition of NF-kappaB activity is a mechanism by which PPAR-gamma agonists improve insulin sensitivity in vivo and that adipocyte NF-kappaB is a potential therapeutic target for obesity-linked type 2 diabetes. PMID: 12732648 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: DNA Cell Biol. 2003 Feb;22(2):131-9. Regulatory effect of SOCS on NF-kappaB activity in murine monocytes/macrophages. Park SH, Kim KE, Hwang HY, Kim TY. Department of Dermatology, The Catholic Research Institutes of Medical Science, Seoul, Korea. The suppressor of cytokine signaling (SOCS) group of proteins has been implicated in regulation of various cytokine signaling and in a negative crosstalk between distinct signaling pathways. Interleukin-10 (IL-10) and LPS were known to induce expression of SOCS-3 in neutrophils and monocytes/macrophages. IL-10 was also reported to inhibit a proinflammatory signal-induced NF-kappaB activation in monocytes and peripheral T lymphocytes. The effects of increased SOCS-3 expression upon IL-10 regulation of NF-kappaB activation have not yet been demonstrated. Here we examined the effects of SOCS-3 on NF-kappaB activity. SOCS-3 did not induce any alterations in NF-kappaB activity induced by LPS or TNF-alpha. However, it enhanced RelA-dependent kappaB promoter activity when cotransfected with RelA. Similar results were observed with SOCS-1. In contrast, SOCS-2 did not show any regulatory effects on RelA activity. Analysis of C-terminal truncation mutants of SOCS-1 and SOCS-3 demonstrated that the SOCS box and its N-terminal region, a less well-conserved linker region were important for SOCS-3 activation of RelA. In contrast, the SOCS box itself was critical for SOCS-1 to activate RelA. These results suggest that SOCS proteins can enhance the effects of NF-kappaB/Rel proteins, and therefore, further modulate immune and inflammatory responses. PMID: 12713738 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: J Immunol. 2003 May 1;170(9):4489-96. Distinct pathways for NF-kappa B regulation are associated with aberrant macrophage IL-12 production in lupus- and diabetes-prone mouse strains. Liu J, Beller DI. Arthritis Section, Evans Department of Medicine and Clinical Research, Boston University Medical Campus, Boston, MA 02118, USA. One characteristic of mice prone to a variety of autoimmune diseases is the aberrant regulation of cytokine production by macrophages (Mphi), noted in cells isolated well before the onset of disease. Strikingly, the pattern of IL-12 dysregulation, in particular, is consistent with the nature of the autoimmune disease that will develop in each strain, i.e., elevated in mice prone to Th1-mediated organ-specific disease (nonobese diabetic (NOD) and SJL mice) and reduced in lupus-prone strains (MRL/+ and NZB/W). Mechanistically, the abnormal regulation of IL-12 in these strains was found to be strictly associated with novel patterns of Rel binding in vitro to the unique NF-kappaB site in the IL-12 p40 promoter. In this study, we report several new findings related to these Rel-kappaB interactions. Evaluation of the p40 NF-kappaB site in vivo, assessed by chromatin immunoprecipitation, revealed Rel usage patterns similar to those found in vitro using EMSA, with preferential association of the p40 kappaB site with c-Rel in NOD Mphi but with p50 in NZB/W Mphi. Moreover, blocking c-Rel in primary Mphi, using short interfering RNA, selectively blocked IL-12 production and normalized the minimal, residual IL-12 levels. Nuclear extracts from NOD Mphi were characterized by c-Rel hyperphosphorylation, and dephosphorylation of nuclear proteins completely blocked binding to the kappaB site. In contrast, elevated IkappaB appears to be a likely mechanism accounting for the reduced nuclear c-Rel levels noted in NZB/W Mphi. Alterations in NF-kappaB metabolism thus appear to define a pathway regulating intrinsic IL-12 defects in both diabetes- and lupus-prone strains. PMID: 12707325 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: J Immunol. 2003 Apr 15;170(8):3963-70. Cross-linking of CD32 induces maturation of human monocyte-derived dendritic cells via NF-kappa B signaling pathway. Banki Z, Kacani L, Mullauer B, Wilflingseder D, Obermoser G, Niederegger H, Schennach H, Sprinzl GM, Sepp N, Erdei A, Dierich MP, Stoiber H. Institute of Hygiene and Social Medicine, Leopold-Franzens University and Ludwig Boltzmann Institute for AIDS Research, Innsbruck, Austria. Dendritic cells (DC) represent a unique set of APCs that initiate immune responses through priming of naive T cells. Maturation of DC is a crucial step during Ag presentation and can be induced by triggering a broad spectrum of DC surface receptors. Although human DC express several receptors for the Fc portion of IgG which were described to play an important role in Ag internalization, little is known about the effects of IgG or immune complexes on DC maturation. In this study, we show that cross-linking of FcgammaR-type II (CD32) with immobilized IgG (imIgG) can induce maturation of human monocyte-derived DC via the NF-kappaB signaling pathway. IgG-mediated maturation was accompanied by a moderate increase of IL-10 secretion, whereas no IL-12 production was observed. Involvement of CD32 was further supported by experiments with the anti-CD32 mAb, which blocked IgG-triggered DC maturation and cytokine secretion significantly. Furthermore, DC cultivated in the presence of imIgG induced allogeneic T cell proliferation. Because this imIgG-induced maturation was considerably impaired in monocyte-derived DC from systemic lupus erythematosus patients, we suggest that DC, which matured in the presence of immune complexes, may contribute to prevention of pathological immune responses. PMID: 12682223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: J Biol Chem. 2003 Jul 18;278(29):26333-41. Epub 2003 Apr 16. BRCA1 augments transcription by the NF-kappaB transcription factor by binding to the Rel domain of the p65/RelA subunit. Benezra M, Chevallier N, Morrison DJ, MacLachlan TK, El-Deiry WS, Licht JD. Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA. BRCA1 is a tumor suppressor gene mutated in cases of hereditary breast and ovarian cancer. BRCA1 protein is involved in apoptosis and growth/tumor suppression. In this study, we present evidence that p65/RelA, one of the two subunits of the transcription factor NF-kappaB, binds to the BRCA1 protein. Treatment of 293T cells with the cytokine tumor necrosis factor-alpha induces an interaction between endogenous p65/RelA and BRCA1. GST-protein affinity assay experiments reveal that the Rel homology domain of the p65/RelA subunit of NF-kappaB interacts with multiple sites within the N-terminal region of BRCA1. Transient transfection of BRCA1 significantly enhances the ability of the tumor necrosis factor-alpha or interleukin-1beta to activate transcription from the promoters of NF-kappaB target genes. Mutation of the NF-kappaB-binding sites in the NF-kappaB reporter blocks the effect of BRCA1 on transcription. Also the ability of BRCA1 to activate NF-kappaB target genes is inhibited by a super-stable inhibitor of NF-kappaB and by the chemical inhibitor SN-50. These data indicate that BRCA1 acts as a co-activator with NF-kappaB. In addition, we show that cells infected with an adenovirus expressing BRCA1 up-regulate the endogenous expression of NF-kappaB target genes Fas and interferon-beta. Together, this information suggests that BRCA1 may play a role in cell life-death decisions following cell stress by modulation of the activity of NF-kappaB. PMID: 12700228 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Neurosci Lett. 2003 Apr 10;340(2):139-42. Activation of nuclear transcription factor-kappa B is associated with the induction of inhibitory kappa B kinase-beta and involves differential activation of protein kinase C and protein tyrosine kinases during fatal murine cerebral malaria. Kumar KA, Rajgopal Y, Pillai U, Babu PP. Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. The levels of nuclear transcription factor-kappa B (NF-kappaB) subunits p65 and p50 and its associated kinase, inhibitory kappa B kinase (IKK) alpha and beta were monitored in cytosolic and nuclear fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Since protein kinase C (PKC) and protein tyrosine kinases (PTK) are known to collaborately regulate the NF-kappaB activation, we also studied the activity of these two kinases in cytosol and membrane fraction. In parallel, the levels of two PKC isoforms (alpha and delta) and tyrosine phosphorylated proteins were monitored to correlate the observed changes in the activity. Our results underscore the involvement of IKK-beta as an essential mediator of NF-kappaB activation as evinced by the nuclear translocation of p65 and p50 during CM pathology. Additional findings confirm altered activity and levels of PKC and enhanced activation of PTK and tyrosine phosphorylation of proteins during CM pathology. These signaling intricacies involving an interplay between rel family (NF-kappaB) of transcription factors, PKC and PTK may serve as an important cue in understanding the possible continuation of the post receptor signaling events associated with tumor necrosis factor-alpha induction during FMCM pathology. Copyright 2003 Elsevier Science Ireland Ltd. PMID: 12668256 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Immunology. 2003 Apr;108(4):539-47. Overexpressed nuclear factor-kappaB can participate in endogenous C-reactive protein induction, and enhances the effects of C/EBPbeta and signal transducer and activator of transcription-3. Agrawal A, Cha-Molstad H, Samols D, Kushner I. Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA. C-reactive protein (CRP), the prototypical human acute phase protein, is produced primarily by hepatocytes. Its expression is modestly induced by interleukin (IL)-6 in Hep3B cells while IL-1, which alone has no effect, synergistically enhances the effects of IL-6. In previous studies of the proximal CRP promoter, we found that signal transducer and activator of transcription-3 (STAT3) and C/EBPbeta -mediated IL-6-induced transcription and that Rel p50 acted synergistically with C/EBPbeta, in the absence of p65, to enhance CRP transcription. Neither a requirement nor a binding site for the classic nuclear factor (NF)-kappaB heterodimer p50/p65 were found. The current studies were undertaken to determine whether similar novel transcription factor interactions might regulate the endogenous CRP gene. Transiently overexpressed p50 or p65 induced CRP mRNA accumulation in Hep3B cells. The heterodimer p50/p65 was markedly more effective than p50 or p65 homodimers. Co-overexpression of p50 or p65 with C/EBPbeta or STAT3 synergistically enhanced CRP expression. Maximal expression was observed with overexpression of all four transcription factors; comparable effects were observed with IL-1beta treatment of cells overexpressing STAT3 + C/EBPbeta. Data from the Human Genome Project revealed 13 potential kappaB sites in the first 4000 bases of the CRP promoter, only one of which, centred at -2652, bound nuclear p50/p65 heterodimer activated by IL-1beta. Our findings indicate that classical NF-kappaB activation can participate in endogenous CRP induction, and that activated NF-kappaB may synergistically enhance the effects of C/EBPbeta and STAT3. They raise the possibility, not as yet established, that NF-kappaB activation may be responsible for the synergistic effect of IL-1beta on IL-6-induced CRP expression. PMID: 12667216 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: J Immunol. 2003 Apr 1;170(7):3724-31. c-Rel is required for chromatin remodeling across the IL-2 gene promoter. Rao S, Gerondakis S, Woltring D, Shannon MF. Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, Canberra, Australia. IL-2 gene transcription occurs in an activation-dependent manner in T cells responding to TCR and CD28 activation. One of the critical events leading to increased IL-2 transcription is an alteration in chromatin structure across the 300-bp promoter region of the gene. We initially showed that IL-2 gene transcription in CD4(+) primary T cells is dependent on the NF-kappaB family member, c-Rel, but not RelA. We found that c-Rel is essential for global changes in chromatin structure across the 300-bp IL-2 promoter in response to CD3/CD28 in primary CD4(+) T cells, but not in response to pharmacological signals, paralleling the requirement for c-Rel in IL-2 mRNA and protein accumulation. Interestingly, measurement of activation-induced localized accessibility changes using restriction enzyme digestion revealed that accessibility close to the c-Rel binding site in the CD28RR region of the promoter is specifically dependent on c-Rel. In contrast, restriction enzyme sites located at a distance from the CD28RR behave independently of c-Rel. These results suggest a nonredundant role for c-Rel in generating a correctly remodeled chromatin state across the IL-2 promoter and imply that the strength of the signal determines the requirement for c-Rel. PMID: 12646638 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: J Biol Chem. 2003 May 9;278(19):17210-7. Epub 2003 Mar 4. p47phox participates in activation of RelA in endothelial cells. Gu Y, Xu YC, Wu RF, Nwariaku FE, Souza RF, Flores SC, Terada LS. Department of Internal Medicine, University of Texas Southwestern, Dallas, Texas 75216, USA. Activation of endothelial cell NF-kappaB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-kappaB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-kappaB activation by IL-1, was associated with the NADPH oxidase adapter protein p47(phox) in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47(phox) synergized with IL-1beta in the activation of an artificial kappaB-luciferase reporter and specifically augmented IL-1beta-induced RelA transactivation activity. p47(phox) overexpression also greatly increased IL-1beta-stimulated RelA phosphorylation, whereas it had no effect on I-kappaB degradation or on RelA nuclear translocation or kappaB binding. The tandem SH3 domains of p47(phox) were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47(phox) and RelA, and ectopic expression of RelA-PR abrogated IL-1beta-induced transactivation of the NF-kappaB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1beta-induced E-selectin promoter activation, suggesting that p47(phox) facilitates NF-kappaB activation through linkage with the NADPH oxidase. IL-1beta rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1beta signaling pathway leading to RelA activation. PMID: 12618429 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Cancer Res. 2003 Mar 1;63(5):1059-66. Differential roles of RelA (p65) and c-Rel subunits of nuclear factor kappa B in tumor necrosis factor-related apoptosis-inducing ligand signaling. Chen X, Kandasamy K, Srivastava RK. Department of Pharmaceutical Sciences, Molecular and Cellular Biology Program, Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland 21201-1180, USA. Apo-2L/TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis through engagement of the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). The transcription factor nuclear factor (NF)-kappa B regulates the expression of genes involved in cancer cell invasion, metastasis, and resistance to chemotherapy. In normal unstimulated cells, NF-kappa B is maintained in the cytoplasm with its inhibitor protein I kappa B, whereas in cancer cells, NF-kappa B is in the nucleus and constitutively activates target genes. To understand the function of NF-kappa B in TRAIL-induced apoptosis, we have analyzed the specific roles of NF-kappa B subunits. Overexpression of a transdominant-negative mutant of the inhibitory protein I kappa B alpha results in down-regulation of constitutively active NF-kappa B, induction of DR5, and tumor necrosis factor receptor (TNFR) 1-associated death domain expression and enhancement of TRAIL sensitivity. Overexpression of RelA or a transcriptional-deficient mutant of c-Rel inhibits TRAIL-induced apoptosis. Depletion of RelA in mouse embryonic fibroblasts increases cytokine-induced apoptosis, whereas depletion of c-Rel blocks this process. Overexpression of RelA subunit inhibits caspase-8 and DR4 and DR5 expression and enhances expression of cIAP1 and c-IAP2 after TRAIL treatment. By comparison, overexpression of c-Rel enhances DR4, DR5, and Bcl-X(s) and inhibits cIAP1, cIAP2, and survivin after TRAIL treatment. These results suggest that the RelA subunit acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2. The dual function of NF-kappa B, as an inhibitor or activator of apoptosis, depends on the relative levels of RelA and c-Rel subunits. Thus, NF-kappa B activity may play an important role in tumor progression, and down-regulation of RelA or up-regulation of c-Rel represents a possible therapeutic target for the treatment of cancer. PMID: 12615723 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Mol Cell Biol. 2003 Feb;23(4):1418-27. Regulation of c-Rel nuclear localization by binding of Ca2+/calmodulin. Antonsson A, Hughes K, Edin S, Grundstrom T. Department of Molecular Biology, Umea University, 901 87 Umea, Sweden. The NF-kappa B/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory I kappa B proteins sequester NF-kappa B/Rel in the cytoplasm. Cellular stimulation results in the degradation of I kappa B and modification of NF-kappa B/Rel proteins, allowing NF-kappa B/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca(2+) binding protein that serves as a key mediator of intracellular Ca(2+) signals. Here we report that two members of the NF-kappa B/Rel family, c-Rel and RelA, interact directly with Ca(2+)-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of I kappa B. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca(2+) signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from I kappa B and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-kappa B/Rel proteins following stimulation. PMID: 12556500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: J Bacteriol. 2003 Feb;185(3):957-65. Characterization of the stringent response and rel(Bbu) expression in Borrelia burgdorferi. Bugrysheva J, Dobrikova EY, Sartakova ML, Caimano MJ, Daniels TJ, Radolf JD, Godfrey HP, Cabello FC. Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA. The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (rel(Bbu)) was produced by B31 as by N40 during in vitro growth (2 +/- 0.5 and 4 +/- 0.8 fg of rel(Bbu) mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 rel(Bbu) mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in rel(Bbu) mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel(Bsu) were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel(Bbu) promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel(Bbu) ATG start codon and identical sigma(70)-like promoters were identified by primer extension and sequencing analysis. rel(Bbu) complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel(Bbu) mRNA. These results indicate that rel(Bbu) is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control. PMID: 12533471 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: EMBO J. 2003 Jan 2;22(1):121-30. RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF. Yilmaz ZB, Weih DS, Sivakumar V, Weih F. Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, D-76021 Karlsruhe, Germany. Targeted disruption of the Rel/NF-kappaB family members NF-kappaB2, encoding p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid tissues. Here, we report that development of Peyer's patch (PP)-organizing centers is impaired in both NF-kappaB2- and RelB-deficient animals. IL-7-induced expression of lymphotoxin (LT) in intestinal cells, a crucial step in PP development, is not impaired in RelB-deficient embryos. LTbeta receptor (LTbetaR)-deficient mice also lack PPs, and we demonstrate that LTbetaR signaling induces p52-RelB and classical p50-RelA heterodimers, while tumor necrosis factor (TNF) activates only RelA. LTbetaR-induced binding of p52-RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by the NF-kappaB-inducing kinase (NIK) and the IkappaB kinase (IKK) complex subunit IKKalpha, but not IKKbeta or IKKgamma. Activation of RelA requires all three IKK subunits, but is independent of NIK. Finally, we show that TNF increases p100 levels, resulting in the specific inhibition of RelB DNA binding via the C-terminus of p100. Our data indicate an important role of p52-RelB heterodimers in lymphoid organ development downstream of LTbetaR, NIK and IKKalpha. PMID: 12505990 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Ai Zheng. 2002 Jun;21(6):663-7. [Expression of NF-kappa B in human bladder cancer and its clinical significance] [Article in Chinese] Xie DH, Tang XD, Xia SJ, Tan JM, Wang XH, Cai Y. Department of Urology, First People's Hospital of Shanghai, Shanghai 200080, P. R. China. xiedh@online.sh.cn BACKGROUND & OBJECTIVE: Studies on solid cancer(such as breast cancer, hepatocellular cancer, pancreatic cancer) indicated that the abnormal expression of nuclear transcription factor Kappa B (NF-kappa B) regulates angiogenesis and cyclin-related genes. This study was designed to investigate the expression differences of NF-kappa B and its regulated genes between human primary transitional cell carcinoma(TCCs) of bladder and non-cancer bladder mucosa and its clinical significance. METHODS: Forty-three frozen sections including 30 bladder cancer and 13 non-cancer bladder mucosa were subjected to immunohistochemistry and nucleus staining for determining levels of NF-kappa B family and I kappa B alpha; Five paired cancer and non-cancer specimens were subjected to Western blot for analysis p65, an important subtype of NF-kappa B; Thirteen paired specimens were subjected to RT-PCR for determination mRNA levels of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8. RESULTS: Expressions of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8 mRNAs in bladder cancer were higher than that in non-cancer bladder mucosa (P < 0.01, P < 0.05, P < 0.01, P < 0.01, P < 0.05, P < 0.05, P < 0.05 and P < 0.05, respectively). Nucelus stainings of p50, p52, p65, c-Rel, RelB were also stronger in bladder cancer(P < 0.01, P < 0.01, P < 0.01, P < 0.01 and P < 0.01, respectively). Moreover, p65 was expressed more in cancer tissue than that in non-cancer mucosa evidenced by Western blot. In bladder cancer, the ranking score differences of p52, p65, c-Rel protein between lymphatic metastasis group and non-lymphatic metastasis group were statistically significant (P < 0.01, P < 0.01, P < 0.05, respectively). CONCLUSIONS: Compared to noncancer bladder mucosa, expressions of NF-kappa B family and its regulated genes in bladder cancer are markedly higher. NF-kappa B may be related to lymphatic metastasis. PMID: 12452071 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: J Immunol. 2002 Dec 1;169(11):6236-43. NF-kappaB regulates the expression of the human complement receptor 2 gene. Tolnay M, Vereshchagina LA, Tsokos GC. Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. mtolnay@hotmail.com CR2 is a key regulator of the B cell response to Ag. Here we show that NF-kappaB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-kappaB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-kappaB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-kappaB p50 and p65. Overexpression of NF-kappaB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-kappaB in regulating promoter activity. Importantly, mutation of the NF-kappaB element or the X box/E box rendered the promoter unresponsive to NF-kappaB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-kappaB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-kappaB element or the X box/E box. Finally, stimuli that activate NF-kappaB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-kappaB signaling pathway enhances the expression of the CR2 gene, as a result of NF-kappaB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface. PMID: 12444129 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Int Immunopharmacol. 2002 Oct;2(11):1567-83. Inhibition of glutathione-related enzymes augments LPS-mediated cytokine biosynthesis: involvement of an IkappaB/NF-kappaB-sensitive pathway in the alveolar epithelium. Haddad JJ, Safieh-Garabedian B, Saade NE, Lauterbach R. Department of Anesthesia and Perioperative Care, University of California at San Francisco, School of Medicine, 94143-0542, USA. haddadj@anesthesia.ucsf.edu The regulation of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokine biosynthesis by reduction-oxidation (redox)-sensitive enzymes involved in maintaining intracellular glutathione homeostasis was investigated in fetal alveolar type II epithelial cells (fATII). Inhibition of glutathione-oxidized disulfide reductase, which recycles GSSG --> 2GSH, by the action of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) augmented LPS-dependent secretion of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. BCNU increased [GSSG] concentration at the expense of [GSH], thereby favoring oxidation equilibrium. Inhibition of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of GSH, by the action of L-buthionine-(S,R)-sulfoximine (BSO), potentiated LPS-induced IL-1beta, IL-6 and TNF-alpha production. Similar to BCNU, BSO depleted [GSH] and induced the accumulation of [GSSG]. BCNU and BSO reduced LPS-mediated phosphorylation of inhibitory-kappaB (IkappaB-alpha), allowing its cytosolic accumulation. This effect was associated with the inhibition of the nuclear translocation of selective nuclear factor (NF)-kappaB subunits: NF-kappaB1 (p50), RelA (p65), RelB (p68) and c-Rel (p75), but not NF-kappaB2 (p52). BCNU and BSO reduced LPS-induced NF-kappaB activation as determined by the electrophoretic mobility shift DNA-binding assay. Analytical analysis of the effect of modulating the dynamic redox ratio ([GSH]+[GSSG])/[GSSG] revealed a novel role for GSSG as a disulfhydryl compound which mediates an inhibitory effect on NF-kappaB activation. It is concluded that selective modulation of redox-sensitive enzymes has an immunopharmacological potential in regulating pro-inflammatory cytokines and that the TkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling. PMID: 12433058 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Clin Cancer Res. 2002 Nov;8(11):3561-9. Adenovirus vector-mediated overexpression of a truncated form of the p65 nuclear factor kappa B cDNA in dendritic cells enhances their function resulting in immune-mediated suppression of preexisting murine tumors. Lee JM, Mahtabifard A, Yamada R, Crystal RG, Korst RJ. Thoracic Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. PURPOSE: The purpose of this study was to evaluate the effect of the Rel homology domain (RHD) of the transcription factor, nuclear factor kappa B (NF kappa B), on proinflammatory gene expression in bone marrow-derived dendritic cells (BMDCs). EXPERIMENTAL DESIGN: We used an adenovirus vector encoding only the RHD of the NF kappa B (p65 family member) cDNA (AdRHD) to transduce murine BMDCs ex vivo. Endpoints measured included BMDC expression of activation markers, cytokine secretion, peptide antigen presentation, and the ability of these transduced cells to induce antitumor immunity in vivo. RESULTS: AdRHD-transduced BMDCs secreted higher levels of the cytokines interleukin (IL) 1 beta, IL-6, and IL-12 (p40) compared with sham-transduced BMDCs or those transduced with an empty vector. AdRHD induced heightened surface expression of the activation markers CD40, B7.1, B7.2, and MHC class II on BMDCs, and these cells were able to present a peptide antigen to a T-lymphocyte hybridoma more efficiently than controls in vitro. Growth of syngeneic, established tumors (CT26 and B16.F10) was inhibited, and survival was prolonged in the mice that received intratumoral AdRHD-modified BMDCs compared with controls. Splenocytes from CT26 tumor-bearing animals that received intratumoral AdRHD-modified BMDCs were able to lyse CT26 target cells more efficiently than controls. Similar experiments using host mice harboring targeted mutations in CD4 and CD8, as well as BMDCs from mice lacking MHC class I, MHC class II, or IL-12 revealed that this tumor immunity was dependent on the presence of CD4+ and CD8+ cells in the tumor-bearing host, as well as MHC class I, MHC class II, and IL-12 expression by the administered BMDCs. Furthermore, induction of IL-12 (p40) expression by AdRHD was completely abrogated in BMDCs lacking the c-Rel NF kappa B family member. CONCLUSIONS: We made the following conclusions: (a) gene transfer-mediated overexpression of the RHD of NF kappa B activates BMDCs; (b) AdRHD-transduced BMDCs induce antitumor immunity when administered intratumorally, an effect mediated by both the CD4+ T cell/MHC class II and the CD8+ T cell/MHC class I pathways, as well as IL-12; and (c) IL-12 (p40) up-regulation by the RHD transgene in BMDCs is dependent on the presence of the c-Rel NF kappa B family member. PMID: 12429647 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: J Infect Dis. 2002 Nov 1;186(9):1199-206. Epub 2002 Oct 11. Respiratory syncytial virus-induced activation of nuclear factor-kappaB in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways. Haeberle HA, Takizawa R, Casola A, Brasier AR, Dieterich HJ, Van Rooijen N, Gatalica Z, Garofalo RP. Department of Pediatrics, The University of Texas Medical Branch, Galveston, Texas 77555, USA. helene.haeberle@uni-tuebingen.de The transcription factor nuclear factor (NF)-kappaB controls the expression of numerous respiratory syncytial virus (RSV)-inducible inflammatory and immunomodulatory genes. Using a BALB/c mouse model, the present article shows that RSV potently and specifically activates NF-kappaB in vivo, a process that involves nuclear translocation of the subunits RelA, p50, and c-Rel in the lung. By depletion of alveolar macrophages (AMs) in BALB/c mice and use of C3H/HeJ mice lacking a functional Toll-like receptor (TLR)-4 signaling pathway, we demonstrate the existence of distinct but sequentially integrated RSV-inducible early NF-kappaB responses in the lung. The first response occurs early after RSV inoculation, is AM and TLR4 dependent, and is viral replication independent, whereas the second response involves epithelial cells and/or inflammatory cells, is TLR4 independent, and requires viral replication. NF-kappaB may be considered a central activator of not only inflammatory but also innate immune responses to RSV. PMID: 12402188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Mol Genet Genomics. 2002 Oct;268(2):179-89. Epub 2002 Aug 15. A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2). Xu J, Tozawa Y, Lai C, Hayashi H, Ochi K. National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp. PMID: 12395192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: J Leukoc Biol. 2002 Oct;72(4):657-67. FcgammaR cross-linking mediates NF-kappaB activation, reduced antigen presentation capacity, and decreased IL-12 production in monocytes without modulation of myeloid dendritic cell development. Drechsler Y, Chavan S, Catalano D, Mandrekar P, Szabo G. Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01605, USA. Stimulation of monocytes (MO) through receptors for the Fc region of immunoglobulin G (FcgammaR) activates a variety of responses, including phagocytosis, antibody (Ab)-dependent cellular cytotoxicity, and production of cytokines. We previously reported that the MO subpopulation that expresses FcgammaR in high density produces high levels of tumor necrosis factor alpha (TNF-alpha) compared with FcgammaR-negative MO. Here, we show that cross-linking MO via FcgammaRI or FcgammaRII but not via FcgammaRIII activates nuclear regulatory factor-kappaB (NF-kappaB), a transcription factor involved in regulation of TNF-alpha. NF-kappaB activation peaked at 2.75 h after FcgammaRI cross-linking, involved p65 and p50 (heterodimer) and not c-rel-containing NF-kappaB complexes, and was mediated via IkappaB degradation. Cross-linking FcgammaRI, -II, as well as -III inhibited interleukin (IL)-12 (p70) production in MO, whether stimulated with Staphylococcal enterotoxin B (P<0.02) or lipopolysaccharide (P<0.02). Inhibition of IL-12 by FcgammaR cross-linking was not mediated by TNF-alpha, as the presence of an anti-TNF-alpha Ab could not restore the reduced IL-12 production. Decreased IL-12 production correlated with reduced antigen presentation capacity (P<0.01) in the FcgammaR-cross-linked MO. Blood MO can give rise to myeloid dendritic cells (DC). FcgammaR cross-linking did not modulate in vitro maturation of MO to fully functional myeloid DC. Allostimulatory capacity in mixed leukocyte reaction and DC marker expression (CDla, CD80, CD86) was not different between control and FcgammaRI cross-linked DC. These results suggest that signals mediated via FcgammaRI, -II, and -III have overlapping yet distinct effects on MO, which are likely to be involved in the fine-tuning of the immune responses to various stimuli. PMID: 12377934 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: J Biol Chem. 2003 Jan 24;278(4):2169-76. Epub 2002 Oct 7. Guanine nucleotides guanosine 5'-diphosphate 3'-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis. Inaoka T, Takahashi K, Ohnishi-Kameyama M, Yoshida M, Ochi K. Microbial Function Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan. We found that a polycistronic operon (ywfBCDEFG) and a monocistronic gene (ywfH) are required for the biosynthesis of bacilysin in Bacillus subtilis. The disruption of these genes by plasmid integration caused loss of the ability to produce bacilysin, accompanied by a lack of bacilysin synthetase activity in the crude extract. We investigated the regulatory mechanism for bacilysin biosynthesis using the transcriptional lacZ fusion system. The transcription of these genes was found to be induced at the transition from exponential to stationary phase. Induction of transcription was accelerated by depleting a required amino acid, which was done by transferring the wild-type (rel(+)) cells to an amino acid-limited medium. In contrast, no enhancement of the gene expression was detected in relA mutant cells. In wild-type (rel(+)) cells, a forced reduction of intracellular GTP, brought about by addition of decoyinine, which is a GMP synthetase inhibitor, enhanced the expression of both the ywfBCDEFG operon and the ywfH gene, resulting in a 2.5-fold increase in bacilysin production. Disruption of the codY gene, which regulates stationary phase genes by detecting the level of GTP, also induced transcription of these genes. In contrast, the expression of ywfBCDEFG in relA cells was not activated either by decoyinine addition or codY disruption, although the expression of ywfH was induced. Moreover, the codY disruption resulted in an increase of bacilysin production only in rel(+) cells. These results indicate that guanosine 5'-diphosphate 3'-diphosphate (ppGpp) plays a crucial role in transcription of the ywfBCDEFG operon and that the transcription of these genes are dependent upon the level of intracellular GTP which is transmitted as a signal via the CodY-mediated repression system. We propose that, unlike antibiotic production in Streptomyces spp., bacilysin production in B. subtilis is controlled by a dual regulation system composed of the guanine nucleotides ppGpp and GTP. PMID: 12372825 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Cancer. 2002 Oct 15;95(8):1696-705. Role of Rel/NF-kappaB transcription factors in apoptosis of human hepatocellular carcinoma cells. Chiao PJ, Na R, Niu J, Sclabas GM, Dong Q, Curley SA. Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. BACKGROUND: Primary hepatocellular carcinoma (HCC) is one of the 10 most common human carcinomas in the world. The mechanism by which HCC cells resist apoptosis induced by various treatment modalities is poorly understood. METHODS: The authors sought to determine whether Rel/NF-kappaB transcription factors play a key role in controlling apoptosis in human HCCcells. We assessed constitutive and inducible activation of NF-kappaB in hepatitis B virus (HBV)-positive (Hep3B) and in hepatitis virus-negative (Chang, HepG2) HCC cells, as well as the role of known inhibitors of NF-kappaB activity. RESULTS: The current study data demonstrate that 1) RelA/NF-kappaB activity is activated constitutively in Hep3B cells, as determined by electrophoretic mobility shift assays; 2) RelA/NF-kappaB reporter gene activity is inhibited specifically by dominant-negative mutants of IkappaB(alpha), IKK1, IKK2, MEKK1, and MEKK3 and it is activated by overexpression of wild-type MEKK3, suggesting that upstream kinase cascades induce phosphorylation of IkappaB(alpha) and activate RelA/NF-kappaB in Hep3B cells; 3) overexpression of the HBV x gene fails to activate NF-kappaB in HepG2 and Chang cell lines; 4) The NF-kappaB-inducible gene, bcl-xl, is overexpressed in Hep3B cells and is inhibited by the proteosome inhibitor PS341, which prevents IkappaBalpha degradation and RelA/NF-kappaB activation; and 5) inhibition of constitutive RelA/NF-kappaB activity by PS341 sensitizes Hep3B cells to doxorubicin-induced apoptosis. CONCLUSIONS: These results are consistent with the role of RelA/NF-kappaB activity in the regulation of apoptosis through activation of its downstream target genes and suggest that signaling pathways that control RelA/NF-kappaB activity may be important targets for novel therapeutic approaches in the treatment of human HCC. Copyright 2002 American Cancer Society. PMID: 12365017 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: Oncogene. 2002 Oct 3;21(44):6819-28. Synergistic and opposing regulation of the stress-responsive gene IEX-1 by p53, c-Myc, and multiple NF-kappaB/rel complexes. Huang YH, Wu JY, Zhang Y, Wu MX. Department of Pathology, Baylor College of Medicine, Houston, Texas, TX 77030, USA. NF-kappaB/rel proteins, tumor suppressor p53, and oncogene c-Myc are critical transcription factors involved in coordinating cellular decision-making events in response to external stimuli. Consensus sequences for binding these three transcription factors are found in the promoter region of IEX-1 (Immediate Early response gene X-1) gene that can either suppress or induce apoptosis in a cell- and stimulus-dependent manner. Utilizing an electrophoretic mobility shift assay (EMSA) and a promoter/reporter assay, we show that the NF-kappaB/rel consensus sequence in the IEX-1 promoter is specifically bound and activated by multiple NF-kappaB/rel complexes in descending order p65-c-rel-->p65-50-->p50-50. Interestingly, NF-kappaB/rel-mediated activation of IEX-1 expression was synergized by p53, but strongly inhibited by c-Myc in a dose-dependent fashion. Moreover, the ability of c-Myc to inhibit IEX-1 expression requires the presence of functional p53, which may partially contribute to the varying effects of p53 on IEX-1 expression in different cells. In support of coordinated regulation of IEX-1 expression by these three transcription factors in vivo, binding of endogenous p53, c-Myc and NF-kappaB/rel proteins, including p50, p65 and c-rel, to the IEX-1 promoter was demonstrated in living cells by chromatin immunoprecipitation using specific antibodies. The study reveals a novel integrative regulation of specific gene expression by NF-kappaB/rel, p53 and c-Myc transcription factors. PMID: 12360408 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: Virology. 2002 Sep 30;301(2):293-304. Activation of cellular and heterologous promoters by the human herpesvirus 8 replication and transcription activator. Roan F, Inoue N, Offermann MK. Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA. The key regulator of the switch from latent to lytic replication of the human herpesvirus 8 (HHV-8; KSHV) is the replication and transcription activator (Rta). The ability of Rta to regulate cellular gene expression was examined by transient transfection into cells that were not infected with HHV-8. Rta induced some, but not all, NF-kappa B-responsive reporters through mechanisms that did not involve activation of classic forms of NF-kappa B. Furthermore, transfection of the NF-kappa B subunit Rel A inhibited the ability of Rta to transactivate some but not all reporters. For example, Rel A inhibited the ability of Rta to transactivate the IL-6 promoter, but only when sequences upstream of the NF-kappa B site were present. The ability of Rel A to inhibit Rta-mediated transactivation was not dependent on a functional NF-kappa B site within the promoter, suggesting an indirect mechanism for inhibition. These studies suggest that Rta expression during lytic reactivation of HHV-8 would lead to expression of some cellular genes, including IL-6, whereas activation of NF-kappa B could inhibit some responses to Rta. PMID: 12359431 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: Oncogene. 2002 Sep 19;21(42):6510-9. The function of multiple IkappaB : NF-kappaB complexes in the resistance of cancer cells to Taxol-induced apoptosis. Dong QG, Sclabas GM, Fujioka S, Schmidt C, Peng B, Wu T, Tsao MS, Evans DB, Abbruzzese JL, McDonnell TJ, Chiao PJ. Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, TX 77030, USA. The Rel/NF-kappaB transcription factors play a key role in the regulation of apoptosis and in tumorigenesis by controlling the expressions of specific genes. To determine the role of the constitutive activity of RelA in tumorigenesis, we generated pancreatic tumor cell lines that express a dominant negative mutant of IkappaBalpha (IkappaBalphaM). In this report, we show that the inhibition of constitutive NF-kappaB activity, either by ectopic expression of IkappaBalphaM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IkappaBalpha proteins, downregulates the expression of bcl-xl. We identified two putative NF-kappaB binding sites (kappaB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-kappaB proteins. p65/p50 heterodimer interacts with kappaB/A site whereas p50/p50 homodimer interacts with kappaB/B. The bcl-xl promoter reporter gene assays reveal that NF-kappaB dependent transcriptional activation is mainly mediated by kappaB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IkappaBalphaM and PS-341 completely abolish NF-kappaB DNA binding activity; however, PS-341, but not ectopic expression of IkappaBalphaM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IkappaBbeta and the re-expression of NF-kappaB regulated bcl-xl gene in these cancer cells as ectopic expression of the bcl-xl gene confers resistance to Taxol-induced apoptosis in PS-341 sensitized cells. These results demonstrate the important function of various NF-kappaB/IkappaB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-kappaB : IkappaBalpha and NF-kappaB : IkappaBbeta complexes are regulated by different upstream activators, and that NF-kappaB plays a key role in pancreatic tumorigenesis. PMID: 12226754 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: J Immunol. 2002 Sep 15;169(6):3329-35. Suppression of monocyte chemoattractant protein 1, but not IL-8, by alprazolam: effect of alprazolam on c-Rel/p65 and c-Rel/p50 binding to the monocyte chemoattractant protein 1 promoter region. Oda T, Ueda A, Shimizu N, Handa H, Kasahara T. Department of Biochemistry, Kyoritsu College of Pharmacy, Tokyo, Japan. oda-ti@kyoritsu-ph.ac.jp Alprazolam is a hypnotic/tranquilizer that has been shown to specifically inhibit the platelet-activating factor (PAF)-induced aggregation of human platelets. The goal of this study was to elucidate whether alprazolam modulates IL-1alpha-initiated responses. For this purpose we investigated the effects of alprazolam on the IL-1alpha-induced production of inflammatory cytokines (IL-8 and monocyte chemoattractant protein 1 (MCP-1)) in a human glioblastoma cell line, T98G, and explored the signaling pathways involved. We found that alprazolam inhibited IL-1alpha-elicited MCP-1 production within a range of 0.1-3 micro M. In contrast, it did not inhibit IL-1alpha-induced IL-8 production. Although NF-kappaB is involved in regulating the IL-1alpha-induced expression of MCP-1 and IL-8, the degradation of IkappaB-alpha stimulated by IL-1alpha was not inhibited by alprazolam. Alprazolam prevented NF-kappaB from binding to the MCP-1 promoter region (the A2 and A1 oligonucleotide probes), but binding of NF-kappaB to IL-8/NF-kappaB was not inhibited. Moreover, alprazolam inhibited c-Rel/p50 binding to the A2 oligonucleotide probe, but not p50/p65 from binding to the IL-8/NF-kappaB site. While AP-1 is involved in regulating the IL-1alpha-induced expression of IL-8, but not MCP-1, alprazolam potentiated the binding of c-Jun/c-Fos to the AP-1 oligonucleotide probe. These results show that the inhibition of IL-1alpha-mediated MCP-1 production by alprazolam is mainly due to inhibition of c-Rel/p65 and c-Rel/p50 binding to the MCP-1 promoter region, since alprazolam did not affect the IL-1alpha-mediated activation of NF-kappaB (p50/p65) or AP-1 (c-Jun/c-Fos) binding to the IL-8 promoter region. In conclusion, a new action of alprazolam was elucidated, as shown in the inhibition of c-Rel/p65- and c-Rel/p50-regulated transcription. PMID: 12218154 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Int Immunol. 2002 Sep;14(9):983-91. Regulation of Ig class switch recombination by NF-kappaB: retroviral expression of RelB in activated B cells inhibits switching to IgG1, but not to IgE. Bhattacharya D, Lee DU, Sha WC. Division of Immunology, Cancer Research Laboratory, Department of Molecular and Cell Biology, University of California, Berkeley 94720-3200, USA. Mutant NF-kappaB-deficient B cells from knockout mice lacking RelA, p105/p50 or the transactivation domain of c-Rel exhibit distinct and selective cell-intrinsic defects in their ability to undergo class switch recombination (CSR) to specific Ig isotypes. This isotype-specific requirement for particular NF-kappaB transcription factors in B cells activated to undergo CSR is intriguing because the NF-kappaB composition in B cells is also highly regulated and can vary significantly depending upon how B cells are activated. These studies prompted us to test by retroviral transduction of normal B cells whether changes in the NF-kappaB composition in activated B cells could modulate cytokine-driven CSR. RelB, RelA, c-Rel, p50 and p52 were first expressed in lipopolysaccharide-activated primary B cells and then induced by cytokine addition to undergo CSR to IgG1, IgE, IgG2a, IgG2b or IgA. Surprisingly, only retroviral expression of RelB altered CSR, resulting in a 3-fold decrease in CSR to IgG1 induced by IL-4. This effect was isotype specific as RelB expression did not affect CSR to IgE within the same culture or to other isotypes tested. The transactivation domain of RelB was required for inhibition of CSR to IgG1. Expression of p50-RelB or p52-RelB dimers joined covalently by a flexible peptide linker also specifically inhibited IgG1 CSR. RelB-mediated inhibition of IgG1 CSR was associated with a decrease in germline gamma1 transcription, but not with changes in proliferation as assayed by CFSE labeling. Thus, RelB complexes can specifically inhibit CSR to IgG1, but not IgE, in activated, primary B cells. PMID: 12202396 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Mol Pharmacol. 2002 Sep;62(3):722-8. 2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses tumor necrosis factor-alpha and anti-CD40-induced activation of NF-kappaB/Rel in dendritic cells: p50 homodimer activation is not affected. Ruby CE, Leid M, Kerkvliet NI. Department of Environmental and Molecular Toxicology, and Environmental Health Science Center, Oregon State University, Corvallis, Oregon 97331, USA. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses many immune responses, both innate and adaptive. Suppression is mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The AhR mediates TCDD toxicity presumably through the alteration of transcriptional events, either by promoting gene expression or potentially by physically interacting with other transcription factors. Another transcription factor, NF-kappaB/Rel, is involved in several signaling pathways in immune cells and is crucial for generating effective immune responses. Dendritic cells (DCs), considered to be the "pacemakers" of the immune system, were recently recognized as targets of TCDD and are also dependent on NF-kappaB/Rel for activation and survival. In these studies, we investigated whether TCDD would alter the activation of NF-kappaB/Rel in DCs. The dendritic cell line DC2.4 was exposed to TCDD before treatment with tumor necrosis factor alpha (TNF-alpha) or anti-CD40, and NF-kappaB/Rel activation was measured by electrophoretic mobility shift assay and immunoblotting. TCDD suppressed the binding of NF-kappaB/Rel to its cognate response element in TNF-alpha- and anti-CD40-treated cells and blocked translocation to the nucleus. The AhR was shown to associate with RelA, after coimmunoprecipitation, and seemed to block its binding to DNA. It is noteworthy that p50 homodimers freely bound to DNA. These results suggest that TCDD may alter the balance between NF-kappaB/Rel heterodimers and transcriptional inhibitory p50 homodimers in DCs, leading to defects in the DCs and suppression of the immune response. PMID: 12181450 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: DNA Cell Biol. 2002 Jul;21(7):491-503. Regulation of the gadd45beta promoter by NF-kappaB. Jin R, De Smaele E, Zazzeroni F, Nguyen DU, Papa S, Jones J, Cox C, Gelinas C, Franzoso G. The Gwen Knapp Center for Lupus and Immunology Research, and The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, Illinois 60637, USA. In addition to coordinating immune and inflammatory responses, NF-kappaB/Rel transcription factors control cell survival. The NF-kappaB antiapoptotic function is crucial to oncogenesis, cancer chemoresistance, and to antagonize tumor necrosis factor (TNF) receptor-induced killing. Recently, we have shown that the suppression of the c-Jun-N-terminal kinase (JNK) cascade is a pivotal protective mechanism by NF-kappaB, and that this suppression involves the upregulation of gadd45beta/myd118. Induction of gadd45beta by stress and cytokines requires NF-kappaB; however, the regulatory mechanisms underlying this induction are not known. Here, we report that, in HeLa cells, the NF-kappaB subunit RelA is sufficient to activate gadd45beta expression, whereas Rel and p50 are not. Activation of gadd45beta by RelA depends on three kappaB elements at positions -447/-438 (kappaB-1), -426/-417 (kappaB-2), and -377/-368 (kappaB-3) of the gadd45beta promoter. Each of these sites binds to NF-kappaB complexes in vitro, and is required for optimal promoter transactivation. The data establish the direct participation of NF-kappaB in the regulation of Gadd45beta, thereby providing important mechanistic insights into the control of apoptosis by the transcription factor. PMID: 12162804 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: Mech Ageing Dev. 2002 May;123(9):1211-27. Age-dependent modulation of NF-kappaB expression in rat adrenal gland. Medicherla R, Leers-Sucheta S, Luo Y, Azhar S. Geriatric Research, Education and Clinical Center (GRECC, 182B), VA Palo Alto Health Care System, 3801 Miranda Ave, Palo Alto, CA 94304, USA. The current studies were initiated to examine the expression and regulation of an oxidative stress-responsive transcription factor, NF-kappa B, in rat adrenals during aging. NF- kappa B DNA-binding activity and expression of constituent proteins (Rel family of proteins and I kappa Bs) was measured in adrenal nuclear and cytoplasmic extracts from young mature (5 month) and old (24 month) Sprague-Dawley rats before and after treatment with LPS; the latter was used to further invoke oxidative stress. Administration of LPS to either young or old rats induced a dramatic activation of NF- kappa B DNA binding activity as assayed by EMSA. NF- kappa B hetero-dimers, RelA/NF- kappa B1 (p65/p50) accounted for the majority of proteins that bound to consensus NF- kappa B sequences in LPS-treated young and old animals. The intensity of DNA binding complexes was significantly reduced in old animals. The age-related decline in the activation of NF- kappa B could not be attributed to an alteration in the composition of constituent subunits or degradation of NF- kappa B inhibitory proteins (I kappa B alpha and I kappa B beta) but rather was due to selective down-regulation of RelA/p65 and NF- kappa B2/p52 proteins. No age-related or LPS-induced changes in the constitutively active transcription factors SP-1 and OCT-1 were detected. These data suggest that aberrancies in the activation of NF- kappa B DNA-binding activity may contribute to the excessive oxidative damage observed in adrenal tissue with aging and may adversely affect cellular processes crucial for intracellular cholesterol transport and steroid hormone production. PMID: 12020944 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: Arterioscler Thromb Vasc Biol. 2002 May 1;22(5):782-7. Long pentraxin PTX3 upregulates tissue factor expression in human endothelial cells: a novel link between vascular inflammation and clotting activation. Napoleone E, Di Santo A, Bastone A, Peri G, Mantovani A, de Gaetano G, Donati MB, Lorenzet R. Antonio Taticchi Unit for Atherosclerosis and Thrombosis, Department of Vascular Medicine and Pharmacology, Istituto Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy. Inflammation is a major contributing factor to atherosclerotic plaque development and ischemic heart disease. PTX3 is a long pentraxin that was recently found to be increased in patients with acute myocardial infarction. Because tissue factor (TF), the in vivo trigger of blood coagulation, plays a dominant role in thrombus formation after plaque rupture, we tested the possibility that PTX3 could modulate TF expression. Human umbilical vein endothelial cells, incubated with endotoxin (lipopolysaccharide) or the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha, expressed TF. The presence of PTX3 increased TF activity and antigen severalfold in a dose-dependent fashion. PTX3 exerted its effect at the transcription level, inasmuch as the increased levels of TF mRNA, mediated by the stimuli, were enhanced in its presence. The increase in mRNA determined by PTX3 originated from an enhanced nuclear binding activity of the transacting factor c-Rel/p65, which was mediated by the agonists and measured by electrophoretic mobility shift assay. The mechanism underlying the increased c-Rel/p65 activity resided in an enhanced degradation of the c-Rel/p65 inhibitory protein IkappaBalpha. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. Our results suggest that PTX3, by increasing TF expression, potentially plays a role in thrombogenesis and ischemic vascular disease. PMID: 12006390 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: J Bacteriol. 2002 Jun;184(11):2878-88. Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis. Mechold U, Murphy H, Brown L, Cashel M. Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2785, USA. Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel(Seq) protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3'-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn(2+)-dependent 3'-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel(Seq) to mediate (p)ppGpp accumulation during amino acid starvation in S. equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of Rel(Seq) with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E. coli by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins. PMID: 12003927 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: J Biol Chem. 2002 Jun 28;277(26):23888-97. Epub 2002 Apr 23. Myotrophin/V-1, a protein up-regulated in the failing human heart and in postnatal cerebellum, converts NFkappa B p50-p65 heterodimers to p50-p50 and p65-p65 homodimers. Knuefermann P, Chen P, Misra A, Shi SP, Abdellatif M, Sivasubramanian N. Winters Center For Heart Failure Research, Molecular Cardiology Unit, Cardiology Section of Department of Medicine, Baylor College of Medicine, Veterans Affairs Medical Center, Houston, Texas 77030, USA. Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of kappaB (NFkappaB)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NFkappaB activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated IkappaBalpha without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NFkappaB proteins in vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NFkappaB activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Adbetagal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NFkappaB activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced kappaB-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NFkappaB activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NFkappaB dimers during this period. PMID: 11971907 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: Oncogene. 2002 Apr 11;21(16):2484-92. Immortalized embryonic mouse fibroblasts lacking the RelA subunit of transcription factor NF-kappaB have a malignantly transformed phenotype. Gapuzan ME, Yufit PV, Gilmore TD. Biology Department, Boston University, 5 Cummington Street, Boston, MA 02215, USA. The RelA transcription factor is part of dimeric complexes, most commonly either p50-RelA (NF-kappaB) heterodimers or RelA homodimers, that control a variety of cellular processes. Immortalized embryonic fibroblasts established from rela knockout mice have previously been shown to be more sensitive to apoptosis induced by tumor necrosis factor (TNF) than are control fibroblasts. In this report, we show that one line of rela-/- fibroblasts has additional phenotypes that distinguish them from control mouse fibroblasts. As compared to normal 3T3 cells, RelA-deficient fibroblasts have a spindled morphology, are less adherent to culture dishes, grow to a higher saturation density, and can form colonies in soft agar. These properties are consistent with a weakly transformed phenotype for rela-/- cells. Furthermore, RelA-deficient fibroblasts can form tumors in immunodeficient mice, but these tumors regress, probably because of the sensitivity of these cells to TNF. The ability of rela-/- fibroblasts to form colonies in soft agar can be reverted by re-expression of wild-type mouse RelA, but not by expression of RelA mutants that cannot form homodimers. There is no clear correlation between the absence of RelA and the levels of expression of other Rel/NF-kappaB family members or adhesion proteins (ICAM-1 and VCAM-1) whose genes have upstream kappaB sites. Taken together, these results suggest that RelA has tumor suppressing activity under some circumstances and that RelA complexes are involved in the control of a variety of cellular properties associated with oncogenesis. PMID: 11971183 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: J Virol. 2002 May;76(10):4928-39. Mutational analysis of the v-Rel dimerization interface reveals a critical role for v-Rel homodimers in transformation. Liss AS, Bose HR Jr. Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712-1095, USA. The v-rel oncogene encoded by reticuloendotheliosis virus strain T is the acutely transforming member of the Rel/NF-kappaB family of transcription factors. In v-Rel-transformed cells, v-Rel exists as homodimers or heterodimers with the endogenous Rel/NF-kappaB proteins c-Rel, NF-kappaB1, NF-kappaB2, and RelA. To examine the contribution of these complexes to v-Rel-mediated transformation, mutations were introduced into the dimerization interface of v-Rel to generate v-Rel mutants with selective dimerization properties. Nine mutants are described in this study that are defective in homodimer and/or heterodimer formation with specific Rel/NF-kappaB family members. Viruses expressing mutants that failed to homodimerize but were able to form heterodimeric complexes were unable to transform splenic lymphocytes in vitro, indicating that the dimerization of v-Rel with endogenously expressed Rel/NF-kappaB proteins is not in itself sufficient for transformation. In addition, two partially transforming mutants were identified that exhibited an impaired ability to form homodimers. Sequence analysis of the proviral DNA from cells transformed by these mutants revealed the presence of multiple secondary mutations in sequences responsible for dimerization and DNA binding. Two of these mutations either enhanced or restored the ability of these proteins to bind DNA as a homodimer. Viruses expressing these proteins transformed cells at levels comparable to or slightly less than v-Rel, suggesting that a threshold level of DNA binding by v-Rel homodimers is required for transformation. PMID: 11967310 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: Cell Signal. 2002 Jul;14(7):633-9. Role of Ca(2+) on TNF-alpha and IL-6 secretion from RBL-2H3 mast cells. Jeong HJ, Hong SH, Lee DJ, Park JH, Kim KS, Kim HM. Department of Oriental Pharmacy, College of Pharmacy, and Korea Institute of Oriental Pharmacy, Wonkwang University, Iksan, 570-749, Chonbuk, South Korea. Ca2+ acts as an important second messenger in mast cells. However, the mechanisms involved in the secretion of inflammatory cytokines from activated mast cells are unknown. In this study, we examined the signaling pathway involved in calcium-related cytokine secretion in a mast cell line, RBL-2H3 cells. We report that treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a chelator of intracellular calcium, can inhibit IgE-stimulated TNF-alpha and IL-6 secretion in a concentration-dependent manner with IC50 values of 0.41 and 0.014 microM, respectively. Maximal inhibition of TNFalpha- and IL-6 secretion was 58.5 +/- 3% and 87 +/- 8% in BAPTA-AM, respectively. BAPTA-AM also completely inhibited the IgE-induced TNF-alpha and IL-6 mRNA levels. In activated RBL-2H3 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus. However, the level of NF-kappaB/Rel A in nucleus was decreased by treatment of BAPTA-AM. In addition, BAPTA-AM completely inhibited the IgE-induced IkappaB kinase beta (IKKbeta) activation and IkappaBalpha phosphorylation. These observations demonstrate that the intracellular Ca2+ may play an important role in IgE-induced TNF-alpha and IL-6 secretion from mast cells via IKKbeta activation. PMID: 11955956 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: J Rheumatol. 2002 Apr;29(4):787-95. Reduction of tumor necrosis factor induced nuclear factor-kappaB nuclear translocation and DNA binding by dexamethasone in human osteoarthritic synovial tissue explants. Lehmann T, Murphy C, Zahra DG, Handel ML. St Vincent's Hospital Clinical School, University of New South Wales, Sydney, Australia. OBJECTIVE: The antiinflammatory effects of glucocorticoids are mediated by several mechanisms, including inhibition of nuclear factor-kappaB (NF-kappaB) nuclear translocation and DNA binding. This mechanism is not evident in some cell types, including endothelial cells and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). We determined the effect of glucocorticoids and tumor necrosis factor (TNF) on nuclear localization and DNA binding of the transcription factor NF-kappaB in osteoarthritic (OA) synovial tissue. METHODS: Explants of synovial tissue from patients undergoing joint replacement surgery for arthritis were placed in culture and treated with dexamethasone 10(-6) M for 18 h and again at 30 min prior to stimulation with TNF for a further 30 min. NF-kappaB and AP-1 DNA binding activities were determined by electrophoretic mobility shift analysis of nuclear extracts prepared from 6 whole tissue explants. Nuclear localization of NF-kappaB was determined by quantitative immunohistochemistry for Rel-A(p65) in thin sections of 5 synovial tissue explants. RESULTS: TNF induced NF-kappaB nuclear translocation and DNA binding in all OA synovial tissue explants, although there were no consistent effects on AP-1 DNA binding. Dexamethasone reduced TNF stimulated nuclear translocation of RelA(p65) in all 5 OA synovial explants analyzed by immunohistochemistry. Dexamethasone partially decreased NF-kappaB DNA binding in 5 of 6 TNF stimulated explants and 4 of 6 unstimulated explants. In cultured rheumatoid arthritis and OA fibroblast-like synoviocytes and Mono Mac 6 cells the effects of dexamethasone on NF-kappaB DNA binding were not evident. CONCLUSION: Dexamethasone partially inhibits TNF induced NF-kappaB DNA binding in human synovial tissue. It is feasible to use explants of intact fresh human synovium as a substrate for the action of antirheumatic drugs targeting a transcription factor. PMID: 11950023 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Appl Environ Microbiol. 2002 Apr;68(4):1541-7. Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene. Okada Y, Makino S, Tobe T, Okada N, Yamazaki S. Department of Veterinary Public Health, The National Institute of Public Health, Tokyo 108-8638, Japan. okada@iph.go.jp Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes. PMID: 11916666 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: BMC Dev Biol. 2002;2:2. Epub 2002 Feb 5. NF-kappa B DNA-binding activity in embryos responding to a teratogen, cyclophosphamide. Torchinsky A, Lishanski L, Wolstein O, Shepshelovich J, Orenstein H, Savion S, Zaslavsky Z, Carp H, Brill A, Dikstein R, Toder V, Fein A. Department of Embryology & Teratology, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel. arkadyt@post.tau.ac.il BACKGROUND: The Rel/NF-kappaB transcription factors have been shown to regulate apoptosis in different cell types, acting as inducers or blockers in a stimuli- and cell type-dependent fashion. One of the Rel/NF-kappaB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFalpha-induced physiological apoptosis. This study assesses whether NF-kappaB may be involved in the embryo's response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstrating differential sensitivity to a reference teratogen, cyclophosphamide, correlates with dysmorphic events induced by the teratogen at the cellular level (excessive apoptosis) and at the organ level (structural anomalies). RESULTS: The embryonic brain and liver were used as target organs. We observed that the Cyclophosphamide-induced excessive apoptosis in the brain, followed by the formation of severe craniofacial structural anomalies, was accompanied by suppression of NF-kappaB DNA-binding activity as well as by a significant and lasting increase in the activity of caspases 3 and 8. However, in the liver, in which cyclophosphamide induced transient apoptosis was not followed by dysmorphogenesis, no suppression of NF-kappaB DNA-binding activity was registered and the level of active caspases 3 and 8 was significantly lower than in the brain. It has also been observed that both the brain and liver became much more sensitive to the CP-induced teratogenic insult if the embryos were exposed to a combined treatment with the teratogen and sodium salicylate that suppressed NF-kappaB DNA-binding activity in these organs. CONCLUSION: The results of this study demonstrate that suppression of NF-kappaB DNA-binding activity in embryos responding to the teratogenic insult may be associated with their decreased resistance to this insult. They also suggest that teratogens may suppress NF-kappaB DNA-binding activity in the embryonic tissues in an organ type- and dose-dependent fashion. PMID: 11893254 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 107: J Biol Chem. 2002 May 17;277(20):17950-61. Epub 2002 Mar 5. Heme oxygenase-1-derived carbon monoxide requires the activation of transcription factor NF-kappa B to protect endothelial cells from tumor necrosis factor-alpha-mediated apoptosis. Brouard S, Berberat PO, Tobiasch E, Seldon MP, Bach FH, Soares MP. Immunobiology Research Center, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. We have shown that carbon monoxide (CO) generated by heme oxygenase-1 (HO-1) protects endothelial cells (EC) from tumor necrosis alpha (TNF-alpha)-mediated apoptosis. This effect relies on the activation of p38 MAPK. We now demonstrate that HO-1/CO requires the activation of the transcription factor NF-kappaB to exert this anti-apoptotic effect. Our data suggest that EC have basal levels of NF-kappaB activity that sustain the expression of NF-kappaB-dependent anti-apoptotic genes required to support the anti-apoptotic effect of HO-1/CO. Over-expression of the inhibitor of NF-kappaB alpha (IkappaBalpha) suppresses the anti-apoptotic action of HO-1/CO. Reconstitution of NF-kappaB activity, by co-expression of IkappaBalpha with different members of the NF-kappaB family, i.e. p65/RelA or p65/RelA plus c-Rel, restores the anti-apoptotic effect of HO-1/CO. Expression of the NF-kappaB family members p65/RelA or p65/RelA with p50 or c-Rel up-regulates the expression of the anti-apoptotic genes A1, A20, c-IAP2, and manganese superoxide dismutase (MnSOD). Inhibition of NF-kappaB activity by over-expression of IkappaBalpha suppresses the expression of some of these anti-apoptotic genes, i.e. c-IAP2. Under inhibition of NF-kappaB, co-expression of some of these anti-apoptotic genes, i.e. c-IAP2 and A1, restores the anti-apoptotic action of HO-1/CO, whereas expression of A20 or MnSOD cannot. The ability of c-IAP2 and/or A1 to restore the anti-apoptotic action of HO-1/CO is abolished when p38 MAPK activation is blocked by over-expression of a p38 MAPK dominant negative mutant. In conclusion, we demonstrate that HO-1/CO cooperates with NF-kappaB-dependent anti-apoptotic genes, i.e. c-IAP2 and A1, to protect EC from TNF-alpha-mediated apoptosis. This effect is dependent on the ability of HO-1/CO to activate the p38 MAPK signal transduction pathway. PMID: 11880364 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 108: Immunity. 2002 Feb;16(2):257-70. Dendritic cell development and survival require distinct NF-kappaB subunits. Ouaaz F, Arron J, Zheng Y, Choi Y, Beg AA. Columbia University, Department of Biological Sciences, 1110 Fairchild Center, 1212 Amsterdam Avenue, New York, NY 10027, USA. Despite the established role of dendritic cells (DCs) in regulating T lymphocyte activation, intracellular mechanisms responsible for controlling DC function are largely undefined. Here, we have studied DCs from mice deficient in the p50, RelA, and cRel subunits of the immunomodulatory NF-kappaB transcription factor. Although DC development and function was normal in mice lacking individual NF-kappaB subunits, development of doubly deficient p50(-/-)RelA(-/-) DCs was significantly impaired. In contrast, DCs from p50(-/-)cRel(-/-) mice developed normally, but CD40L- and TRANCE-induced survival and IL-12 production was abolished. Surprisingly, no significant impairment in MHC and costimulatory molecule expression was seen, despite significantly reduced kappaB site binding activity. These results therefore indicate essential, subunit-specific functions for NF-kappaB proteins in regulating DC development, survival, and cytokine production. PMID: 11869686 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 109: Cell Death Differ. 2002 Mar;9(3):252-63. Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, Birrell GW, Peehl DM, Knox SJ. Department of Radiation Oncology, Stanford University, Stanford, California, CA 94305-5105, USA. The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis. PMID: 11859408 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 110: Thromb Haemost. 2002 Jan;87(1):155-62. Mechanism of resveratrol-mediated suppression of tissue factor gene expression. Pendurthi UR, Meng F, Mackman N, Rao LV. Biomedical Research, The University of Texas Health Center at Tyler, 75708, USA. Tissue factor (TF) is a cell surface receptor for factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade. Inappropriate in vivo expression of TF in vascular cells has been shown to be responsible for thrombotic disorders associated with a variety of pathological conditions, including gram-negative sepsis, cancer and atherosclerosis. A number of epidemiological studies suggest that moderate consumption of red wine provides protective effects against coronary heart disease mortality. Recently, we have shown that resveratrol, a polyphenolic compound found in wine, inhibited the induction of TF expression in endothelial cells and mononuclear cells (Pendurthi UR, Williams JT, Rao LVM. Arterioscler Thromb Vasc Biol 1999: 19: 419-426). In the present study, we examined the mechanism by which resveratrol inhibits the expression of TF in monocytes by using a monocytic cell line, THP-1, as a model cell. Northern blot analysis, gel mobility shift assays and transfection studies with various TF promoter constructs, as well as other transcription regulatory constructs, were used to elucidate the inhibitory mechanism of resveratrol. The data show that resveratrol inhibited lipopolysaccharide (LPS)-induced expression of TF in human monocytes and monocytic cell line, THP-1 in a dose dependent manner. Resveratrol did not significantly alter the binding of various transcription factors involved in TF gene expression to DNA. However, resveratrol suppressed the transcription of cloned human TF promoter. Further experiments revealed that resveratrol reduced kappaB- but not AP-1-driven transcriptional activity. Additional experiments showed that resveratrol suppressed the phosphorylation of p65 and its transactivation. In summary, our results indicate that resveratrol does not inhibit the activation or translocation of NF-kappaB/Rel proteins but inhibits NF-kappaB/Rel-dependent transcription by impairing the transactivation potential of p65. PMID: 11858183 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 111: J Pharmacol Exp Ther. 2002 Feb;300(2):567-76. Immunopharmacological potential of selective phosphodiesterase inhibition. II. Evidence for the involvement of an inhibitory-kappaB/nuclear factor-kappaB-sensitive pathway in alveolar epithelial cells. Haddad JJ, Land SC, Tarnow-Mordi WO, Zembala M, Kowalczyk D, Lauterbach R. Neuroscience Research Laboratory, Department of Anesthesia and Perioperative Care, University of California Medical Center, San Francisco, California 94143, USA. jhaddad@itsa.ucsf.edu In this report we investigated the immunopharmacological role of selective and nonselective phosphodiesterase (PDE) inhibition in regulating the inhibitory-kappaB (IkappaB-alpha)/nuclear factor-kappaB (NF-kappaB) signaling transduction pathway. In fetal alveolar type II epithelial cells, PDE blockade at the level of the diverging cAMP/cGMP pathways differentially regulated the phosphorylation and degradation of IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB. Whereas selective inhibition of PDEs 1, 3, and 4, by the action of 8-methoxymethyl-3-isobutyl-1-methylxanthine, amrinone, and rolipram, respectively, exhibited a tendency to augment the translocation of NF-kappaB(1) (p50), RelA (p65), RelB (p68), and c-Rel (p75), selective blockade of PDE 5, 6, and 9, by the action of 4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline and zaprinast, attenuated lipopolysaccharide-endotoxin (LPS)-mediated NF-kappaB translocation. Pentoxifylline, a nonspecific PDE inhibitor, reversed the excitatory effect of LPS on NF-kappaB subunit nuclear localization, in a dose-dependent manner. Furthermore, analysis of NF-kappaB activation under the same conditions revealed a biphasic effect mediated by LPS. PDEs 1, 3, and 4 inhibition was associated with up-regulating NF-kappaB transcriptional activity. In contrast, blockading the activity of PDEs 5, 6, and 9 negatively attenuated LPS-mediated NF-kappaB activation, similar to the effect of 3,7-dihydro-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6-dione (pentoxifylline). These results indicate that selective and nonselective interference with the control of the dynamic equilibrium of cyclic nucleotides via PDE isoenzyme regulation represents an immunoregulatory mechanism that requires the differential, biphasic targeting of the IkappaB-alpha/NF-kappaB pathway. PMID: 11805218 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 112: J Exp Med. 2002 Jan 21;195(2):233-44. Requirement for the NF-kappaB family member RelA in the development of secondary lymphoid organs. Alcamo E, Hacohen N, Schulte LC, Rennert PD, Hynes RO, Baltimore D. Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. The transcription factor nuclear factor (NF)-kappaB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-kappaB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-kappaB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the mice that are born lack lymph nodes, Peyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell-dependent antigen responses. Analyses of TNFR1/RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs. PMID: 11805150 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 113: J Immunol. 2001 Dec 15;167(12):6827-33. The Src-protein tyrosine kinase Lck is required for IL-1-mediated costimulatory signaling in Th2 cells. al-Ramadi BK, Welte T, Fernandez-Cabezudo MJ, Galadari S, Dittel B, Fu XY, Bothwell AL. Department of Medical Microbiology and Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates. ramadi.b@uaeu.ac.ae Src-protein tyrosine kinases are intimately involved in TCR-initiated signaling in T lymphocytes. One member of this family, Lck, is also involved in CD28-mediated costimulation in Th1 cells. In Th2 lymphocytes, the costimulatory signal can also be provided by the interaction of IL-1 with type I IL-1R (IL-1RI), culminating in the activation of NF-kappaB transcription factors. Proximal steps in the IL-1R pathway, however, remain poorly understood, and there is conflicting evidence as to the importance of tyrosine phosphorylation in IL-1R signaling. We have addressed this issue by examining the ability of IL-1 to costimulate the activation of Lck-deficient Th2 cells. Our data demonstrate that, in the absence of Lck, the IL-1 costimulatory pathway is blocked despite the expression of normal levels of IL-1RI. Moreover, the block is associated with a defective degradation of IkappaB-alpha and an incomplete activation of NF-kappaB heterodimeric complexes. Protein expression of NF-kappaB monomers, including p50, p65, and c-Rel, is equivalent in both wild-type and Lck-deficient Th2 cell clones. Finally, we demonstrate that, in normal Th2 cells, stimulation with IL-1 leads to a rapid induction in tyrosine phosphorylation of several substrates including Lck itself. These findings strongly suggest that Lck is required for signaling in the IL-1 costimulatory pathway in Th2 lymphocytes. PMID: 11739499 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 114: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14328-33. Epub 2001 Nov 20. RelE, a global inhibitor of translation, is activated during nutritional stress. Christensen SK, Mikkelsen M, Pedersen K, Gerdes K. Department of Biochemistry and Molecular Biology, University of Southern Denmark, OU, Campusvej 55, DK-5230 Odense M, Denmark. The stringent response is defined as the physiological changes elicited by amino acid starvation. Many of these changes depend on the regulatory nucleotide ppGpp (guanosine tetraphosphate) synthesized by RelA (ppGpp synthetase I), the relA-encoded protein. The second rel locus of Escherichia coli is called relBE and encodes RelE cytotoxin and RelB antitoxin. RelB counteracts the toxic effect of RelE. In addition, RelB is an autorepressor of relBE transcription. Here we reveal a ppGpp-independent mechanism that reduces the level of translation during amino acid starvation. Artificial overexpression of RelE severely inhibited translation. During amino acid starvation, the presence of relBE caused a significant reduction in the poststarvation level of translation. Concomitantly, relBE transcription was rapidly and strongly induced. Induction of transcription occurred independently of relA and spoT (encoding ppGpp synthetase II), but instead depended on Lon protease. Consistently, Lon was required for degradation of RelB. Replacement of the relBE promoter with a LacI-regulated promoter indicated that strong and ongoing transcription of relBE is required to maintain a proper RelB:RelE ratio during starvation. Thus relBE may be regarded as a previously uncharacterized type of stress-response element that reduces the global level of translation during nutritional stress. PMID: 11717402 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 115: J Immunol. 2001 Dec 1;167(11):6412-20. A novel NF-kappa B/Rel site in intron 1 cooperates with proximal promoter elements to mediate TNF-alpha-induced transcription of the human polymeric Ig receptor. Schjerven H, Brandtzaeg P, Johansen FE. Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, University of Oslo, Rikshospitalet, Oslo, Norway. hilde.schjerven@labmed.uio.no Secretory Abs constitute the first line of specific immune defense at mucosal surfaces. Such Abs are generated by the active transport of polymeric Ig (pIg) across secretory epithelia mediated by the pIgR, also known as transmembrane secretory component (SC). The proinflammatory cytokine TNF-alpha is a key mediator of host responses to infections, and it can stimulate protein synthesis-dependent transcriptional up-regulation of pIgR/SC in the HT-29 intestinal adenocarcinoma cell line. By reporter gene assay we identified a novel TNF-alpha-responsive region located within a 748-bp fragment in intron 1 of the human pIgR/SC gene which depended on an NF-kappaB/Rel site for full responsiveness. EMSAs demonstrated preferential binding of the NF-kappaB/Rel family member p65 (RelA) to this DNA element after TNF-alpha stimulation, with weaker and more delayed binding of p50. Furthermore, the TNF-alpha-responsive region in intron 1 required cooperation with DNA elements located in the proximal promoter region of the gene. Mutational analysis demonstrated that an IFN-stimulated response element near the transcriptional start site in exon 1 was involved in the TNF-alpha responsiveness. Thus, DNA elements located >4 kb apart were found to cooperate in TNF-alpha-induced pIgR/SC up-regulation. The intronic TNF-alpha-responsive enhancer overlapped with a recently identified IL-4-responsive enhancer. Several intronic DNA elements found to be functionally important in the human gene are highly conserved between the human and mouse pIgR/SC genes, suggesting the presence of a conserved cytokine-responsive enhancer region. PMID: 11714807 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 116: J Immunol. 2001 Nov 1;167(9):4948-56. c-Rel is required for the protection of B cells from antigen receptor-mediated, but not Fas-mediated, apoptosis. Owyang AM, Tumang JR, Schram BR, Hsia CY, Behrens TW, Rothstein TL, Liou HC. Division of Immunology, Department of Medicine, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA. The NF-kappaB/Rel transcription factor family has been shown to protect many cell types from apoptotic signals. However, it is not known whether NF-kappaB is required for all survival pathways and whether each NF-kappaB member plays a unique or a redundant role. Here we describe the results of studies on the role of c-Rel in survival. Mature B cells from c-Rel(-/-) mice exhibit defects in survival, including sensitivity to Ag receptor-mediated apoptosis as well as increased sensitivity to ionizing radiation and glucocorticoids. Transgene expression of Bcl-x(L), a c-Rel target gene, rescues c-Rel(-/-) B cells from their survival defects. Thus, c-Rel-dependent survival pathways are crucial for protection from apoptotic signals that target the mitochondrial pathway. Despite a lack of Bcl-x(L), c-Rel(-/-) B cells can still be rescued from Fas-mediated apoptosis via B cell receptor signaling. The Fas apoptosis inhibitor molecule and FLICE inhibitory protein (c-FLIP) proteins are up-regulated normally in c-Rel(-/-) B cells, and these two molecules may play a more physiological role in the Fas pathway. Furthermore, unlike the TNF sensitivity of RelA(-/-) fibroblasts, c-Rel-deficient fibroblasts are refractory to TNF-mediated cell death. Thus, c-Rel is dispensable for protection against death receptor-mediated apoptosis. Taken together, our data suggest that distinct NF-kappaB/Rel members are required for protecting cells from different types of apoptotic signals. PMID: 11673501 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 117: J Biol Chem. 2001 Dec 21;276(51):47828-33. Epub 2001 Oct 8. Protein phosphatase 2A interacts with and directly dephosphorylates RelA. Yang J, Fan GH, Wadzinski BE, Sakurai H, Richmond A. Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175, USA. Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors are key regulators of a variety of genes involved in inflammatory responses, growth, differentiation, apoptosis, and development. There are increasing lines of evidence that NF-kappa B/Rel activity is controlled to a great extent by its phosphorylation state. In this study, we demonstrated that RelA physically associated with protein phosphatase 2A (PP2A) subunit A (PR65). Both the N- and C-terminal regions of RelA were responsible for the PP2A binding. RelA co-immunoprecipitated with PP2A in melanocytes in the absence of stimulation, indicating that RelA forms a signaling complex with PP2A in the cells. RelA was dephosphorylated by a purified PP2A core enzyme, a heterodimer formed by the catalytic subunit of PP2A (PP2Ac) and PR65, in a concentration-dependent manner. Okadaic acid, an inhibitor of PP2A at lower concentration, increased the basal phosphorylation of RelA in melanocytes and blocked the dephosphorylation of RelA after interleukin-1 stimulation. Interestingly, PP2A immunoprecipitated from melanocytes was able to dephosphorylate RelA, whereas PP2A immunoprecipitated from melanoma cell lines exhibited decreased capacity to dephosphorylate RelA in vitro. Moreover, in melanoma cells in which I kappa B kinase activity was inhibited by sulindac to a similar level as in melanocytes, the phosphorylation state of RelA and the relative NF-kappa B activity were still higher than those in normal melanocytes. These data suggest that the constitutive activation of RelA in melanoma cells (Yang, J., and Richmond, A. (2001) Cancer Res. 61, 4901-4909) could be due, at least in part, to the deficiency of PP2A, which exhibits decreased dephosphorylation of NF-kappa B/RelA. PMID: 11591705 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 118: Mol Cell Biol. 2001 Oct;21(20):7065-77. The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Ashburner BP, Westerheide SD, Baldwin AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes. PMID: 11564889 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 119: J Immunol. 2001 Oct 1;167(7):3652-60. STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65. Kawashima T, Murata K, Akira S, Tonozuka Y, Minoshima Y, Feng S, Kumagai H, Tsuruga H, Ikeda Y, Asano S, Nosaka T, Kitamura T. Division of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines PMID: 11564778 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 120: Genetics. 2001 Sep;159(1):189-99. Drosophila immunity: genes on the third chromosome required for the response to bacterial infection. Wu LP, Choe KM, Lu Y, Anderson KV. Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA. We have screened the third chromosome of Drosophila melanogaster for mutations that prevent the normal immune response. We identified mutant lines on the basis of their failure to induce transcription of an antibacterial peptide gene in response to infection or their failure to form melanized clots at the site of wounding. These mutations define 14 genes [immune response deficient (ird) genes] that have distinct roles in the immune response. We have identified the molecular basis of several ird phenotypes. Two genes, scribble and kurtz/modulo, affect the cellular organization of the fat body, the tissue responsible for antimicrobial peptide production. Two ird genes encode components of the signaling pathways that mediate responses to bacterial infection, a Drosophila gene encoding a homolog of I kappa B kinase (DmIkk beta) and Relish, a Rel-family transcription factor. These genetic studies should provide a basis for a comprehensive understanding of the genetic control of immune responses in Drosophila. PMID: 11560896 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 121: J Mol Microbiol Biotechnol. 2001 Oct;3(4):585-600. Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins). Mittenhuber G. Institut fur Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universitat, Greifswald, Germany. Gerhard.Mittenhuber@biologie.uni-greifswald.de In the gram-negative model organism Escherichia coli, the effector molecule of the stringent response, (p)ppGpp, is synthesized by two different enzymes, RelA and SpoT, whereas in the gram-positive model organism Bacillus subtilis only one enzyme named Rel is responsible for this activity. Rel and SpoT also possess (p)ppGpp hydrolase activity. BLAST searches were used to identify orthologous genes in databases. The construction and bootstrapping of phylogenetic trees allowed classification of these orthologs. Four groups could be distinguished: With the exception of Neisseria and Bordetella (beta subdivision), the RelA and SpoT groups are exclusively found in the gamma subdivision of proteobacteria. Two Rel groups representing the actinobacterial and the Bacillus/Clostridium group were also identified. The SpoT proteins are related to the gram positive Rel proteins. RelA proteins carry substitutions in the HD domain (Aravind and Koonin, 1998, TIBS 23: 469-472) responsible for ppGpp degradation. A theory for the evolution of the specialized, paralogous relA and spoT genes is presented: After gene duplication of an ancestral rellike gene, the spoT and relA genes evolved from the duplicated genes. The distribution pattern of the paralogous RelA and SpoT proteins supports a new model of linear bacterial evolution (Gupta, 2000, FEMS Microbiol. Rev. 24: 367-402). This model postulates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. However, two paralogous, closely related genes of Porphyromonas gingivalis (Cytophaga-Flavobacterium-Bacteroides phylum) encoding proteins with functions probably identical to the RelA and SpoT proteins do not fit in this model. Completely sequenced genomes of several obligately parasitic organisms (Treponema pallidum, Chlamydia species, Rickettsia prowazekii) and the obligate aphid symbiont Buchnera sp. APS as well as archaea do not contain rel-like genes but they are present in the Arabidopsis genome. In crosslinking experiments using different analogs of ppGpp as crosslinking reagents and RNA polymerase preparations of Escherichia coli, binding of ppGpp to distinct regions at the C-terminus of the beta subunit (the RpoB gene product) and/or at the N-terminus of the beta subunit (the RpoC gene product) was observed previously. RpoB and RpoC sequences of the species which do not possess a rel like gene do not exhibit specific insertions or deletions in the ppGpp binding regions. PMID: 11545276 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 122: J Mol Cell Cardiol. 2001 Jun;33(6):1223-32. Effect of NF-kappa B Inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes. Bergmann MW, Loser P, Dietz R, von Harsdorf R. Department of Cardiology, Franz Volhard Clinic, Charite, Humboldt University, Berlin, Germany. Heart-specific inhibition of survival pathway gp130 was recently shown to sensitize transgenic mice towards stress stimuli, resulting in rapid onset of cardiac dilatation and heart failure. In order to identify further survival pathways we evaluated the role of transcription factor nuclear factor-kappa B (NF-kappa B) in tumour necrosis factor-alpha (TNF-alpha)-induced apoptosis of cardiomyocytes. TNF-alpha stimulation (10 ng/ml) of both H9c2 cells and primary cardiomyocytes isolated from neonatal Wistar rats resulted in rapid nuclear translocation of NF-kappa B complexes. The NF-kappa B complexes consisted of rel-proteins p50 and p65, as revealed by supershift analysis. Addition of proteasome inhibitor MG132 or adenoviral expression of a truncated I kappa B alpha (I kappa B Delta N) inhibited TNF-alpha-induced NF-kappa B nuclear translocation in a dose-dependent manner. Both neonatal cardiomyocytes and H9c2 cells were resistant to TNF-induced apoptosis. However, specific inhibition of NF-kappa B activation by Ad5-I kappa B alpha Delta N (MOI=50) or MG132 (5 microm) increased apoptosis as measured by subG1-assay (H9c2 cells) and annexin V binding/propidium iodide (neonatal cardiomyocytes, FACS-analysis: 7+/-2% to 26+/-5% annexin V positive/PI negative), respectively. TUNEL-assay double-stained with anti-alpha-sarcomeric actin confirmed apoptosis of neonatal cardiomyocytes. Furthermore, caspase-3 activation was increased by 52+/-7% in neonatal cardiomyocytes after TNF alpha+Ad5-I kappa B alpha Delta N compared to TNF alpha+Ad5-control treatment. Protein levels of hiAP1, hiAP2, x-iAP, bcl-2 and bcl-x(L) were neither downregulated by NF-kappa B inhibition nor upregulated by TNF-alpha stimulation. In summary, cardiomyocytes utilize transcription factor NF-kappa B to activate survival factors in the context of TNF-alpha stimulation. As locally increased levels of TNF-alpha have been detected in heart failure, NF-kappa B activity is essential for cellular homeostasis in the heart. Copyright 2001 Academic Press. PMID: 11444925 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 123: Mol Cell Biol. 2001 Jul;21(14):4837-46. Cell-specific association and shuttling of IkappaBalpha provides a mechanism for nuclear NF-kappaB in B lymphocytes. Tam WF, Wang W, Sen R. Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA. Mature B lymphocytes are unique in containing nuclear Rel proteins prior to cell stimulation. This activity consists largely of p50-c-Rel heterodimers, and its importance for B-cell function is exemplified by reduced B-cell viability in several genetically altered mouse strains. Here we suggest a mechanism for the cell specificity and the subunit composition of constitutive B-cell NF-kappaB based on the observed properties of Rel homo- and heterodimers and IkappaBalpha. We show that c-Rel lacks a nuclear export sequence, making the removal of c-Rel-containing complexes from the nucleus less efficient than removal of p65-containing complexes. Second, the nuclear import potential of p65 and c-Rel homodimers but not p50-associated heterodimers was attenuated when they were complexed to IkappaBalpha, leading to a greater propensity of heterodimers to be nuclear. We propose that subunit composition of B-cell NF-kappaB reflects the inefficient retrieval of p50-c-Rel heterodimers from the nucleus. Cell specificity may be a consequence of c-Rel-IkappaBalpha complexes being present only in mature B cells, which leads to nuclear c-Rel due to IkappaBalpha turnover and shuttling of the complex. PMID: 11416157 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 124: J Biol Chem. 2001 Jul 20;276(29):27203-6. Epub 2001 May 22. Invariant chain induces B cell maturation by activating a TAF(II)105-NF-kappaB-dependent transcription program. Matza D, Wolstein O, Dikstein R, Shachar I. Departments of Immunology and Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Early stages of B cell development occur in the bone marrow, resulting in formation of immature B cells. From there these immature cells migrate to the spleen where they differentiate to mature cells. This final maturation step is crucial for the B cells to become responsive to antigens and to participate in the immune response. Recently, invariant chain (Ii), a major histocompatibility complex class II chaperone, as well as the transcription factors c-Rel and p65/RelA, were found to play a role in the final antigen-independent differentiation stage of B cells in the spleen. In this study, we investigated a possible link between Ii-dependent B cell maturation and the NF-kappaB pathway. Our studies indicate that Ii-induced B cell maturation involves activation of transcription mediated by the NF-kappaB p65/RelA homodimer and requires the B cell-enriched coactivator TBP-associated factor (II)105. PMID: 11371575 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 125: J Biol Chem. 2001 Jul 20;276(29):27322-8. Epub 2001 May 11. Rel/NF-kappaB transcription factors protect against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by up-regulating the TRAIL decoy receptor DcR1. Bernard D, Quatannens B, Vandenbunder B, Abbadie C. Formation de Recherche en Evolution 2353 and Unite Mixte de Recherche 8526 CNRS/Institut Pasteur de Lille/Universite Lille 2, Institut de Biologie de Lille, 1 rue Calmette, 59021 Lille Cedex, France. Rel/nuclear factor (NF)-kappaB transcription factors play a major role in the regulation of programmed cell death. A few anti-apoptotic Rel/NF-kappaB target genes have been characterized; they act either downstream in the apoptotic pathway or upstream, for example at the tumor necrosis factor (TNF) receptor level. We found using DNA arrays, reverse transcription-polymerase chain reaction, and immunofluorescence that Rel/NF-kappaB factors up-regulate DcR1, a receptor for TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family that induces apoptosis in tumor cells. Four related receptors bind TRAIL, two death receptors (DR4 and DR5) that signal apoptosis and two decoy receptors (DcR1 and DcR2) that act as dominant negative inhibitors of TRAIL-mediated apoptosis. DcR1 is devoid of an intracellular domain and is anchored at the cell surface membrane by a glycophospholipid. Our results indicate that overexpression of cRel or activation of endogenous Rel/NF-kappaB factors by TNFalpha in HeLa cells up-regulates DcR1 without changing the expression of DcR2, DR4, and DR5 and makes cells resistant against TRAIL-induced apoptosis. This resistance is a consequence of DcR1 up-regulation, because it was abolished when DcR1 was removed from the cell surface by a phosphatidylinositol phospholipase C. Therefore, Rel/NF-kappaB transcription factors could regulate the sensitivity of cells to TRAIL, by controlling the ratio of TRAIL-decoy to -death receptors. PMID: 11350953 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 126: Microbes Infect. 2001 Apr;3(4):259-65. Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide. Hofer S, Rescigno M, Granucci F, Citterio S, Francolini M, Ricciardi-Castagnoli P. Department of Biotechnology and Bioscience, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B/Rel (NF kappa B/Rel) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines such as IL-1, IL-6, TNF-alpha and cell surface molecules (MHC class I and II, B7.2). In this study, we have compared the activation of five members of the NF-kappa B family, p65, c-Rel, p50, RelB and p52, during DC maturation in response to lipopolysaccharide (LPS) and to Salmonella typhimurium. We have shown that although the translocation of NF-kappa B occurred very early, 30 min after treatment with both S. typhimurium and LPS, bacteria-induced NF-kappa B activation was more pronounced. Four out of five members, i.e. p65, c-Rel, p50 and RelB, were similarly activated upon the two stimuli but with different kinetics. Indeed, we have observed that p65, c-Rel and p50 were translocated early, whereas RelB was translocated later in DC activation. This differential regulation suggests that the various members of NF-kappa B family can mediate distinct functions of DC physiology. PMID: 11334742 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 127: Br J Pharmacol. 2001 May;133(1):49-60. The biphasic immunoregulation of pyrimidylpiperazine (Y-40138) is IL-10 sensitive and requires NF-kappa B targeting in the alveolar epithelium. Haddad JJ, Safieh-Garabedian B, Saade NE, Land SC. Oxygen Signalling Group, Centre for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY. j.j.haddad@dundee.ac.uk 1. Pyrimidylpiperazine (Y-40138), a synthetic derivative of N-[1-(4-([4-(pyrimidin-2-yl)piperazin-1-yl]methyl)phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro- and anti-inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. 2. In alveolar epithelial cells, pre-treatment (30 min) with Y-40138 reduced LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha, an effect paralleled by up-regulating an anti-inflammatory counter-loop mediated through IL-10. 3. This differential regulation of pro- and anti-inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF-kappa B subunits, particularly NF-kappa B(1) (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c-Rel (p75). In addition, Y-40138 blockaded, in a dose-dependent manner, the LPS-induced nuclear activation of NF-kappa B. 4. Analysis of the upstream pathway involved in Y-40138-dependent retardation of LPS-induced NF-kappa B translocation/activation revealed the involvement of an I kappa B-alpha sensitive pathway. Pre-treatment with Y-40138 ameliorated LPS-induced degradation of I kappa B-alpha in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. 5. Recombinant IL-10 (0 -- 10 ng ml(-1)) blockaded, in a dose-dependent manner, LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha. Furthermore, rhIL-10 reduced the DNA binding activity of NF-kappa B. Immunoneutralization of endogenous IL-10 by a polyclonal alpha IL-10 (5 microg ml(-1)) reversed the inhibitory effect of Y-40138 on pro-inflammatory cytokines and partially restored the DNA binding activity of NF-kappa B. 6. These results indicate that Y-40138 mediated dual immunoregulation of pro- and anti-inflammatory cytokines is IL-10 sensitive and mediated through the I kappa B-alpha/NF-kappa B signal transduction pathway. PMID: 11325794 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 128: Mod Pathol. 2001 Apr;14(4):297-310. Characterization of NF-kappaB expression in Hodgkin's disease: inhibition of constitutively expressed NF-kappaB results in spontaneous caspase-independent apoptosis in Hodgkin and Reed-Sternberg cells. Izban KF, Ergin M, Huang Q, Qin JZ, Martinez RL, Schnitzer B, Ni H, Nickoloff BJ, Alkan S. Department of Pathology, Loyola University Medical Center, Maywood, Illinois 60153, USA. Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-kappaB, the precise physiologic and clinical significance of NF-kappaB expression is currently undefined. Expression of active NF-kappaB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-kappaB inhibitors gliotoxin and MG132 and the effect of NF-kappaB inhibition utilizing an adenovirus vector carrying a dominant-negative IkappaBalpha mutant (Ad5IkappaB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin's disease. After chemical NF-kappaB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-kappaB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-kappaB inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD-FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IkappaB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-kappaB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-kappaB in HRS cell survival and resistance to apoptosis. PMID: 11301346 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 129: J Immunol. 2001 Apr 15;166(8):4949-57. NF-kappa B RelA (p65) is essential for TNF-alpha-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death. Zheng Y, Ouaaz F, Bruzzo P, Singh V, Gerondakis S, Beg AA. Department of Biological Sciences, Columbia University, New York, NY 10027, USA. The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-alpha and IFN-gamma or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-alpha and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-kappaB complexes. To determine whether RelA and/or other NF-kappaB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA(-/-), c-Rel(-/-), and p50(-/-) mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-kappaB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-kappaB subunits. PMID: 11290773 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 130: Dev Growth Differ. 2001 Apr;43(2):145-54. Involvement of Rel/NF-kappaB in regulation of ascidian notochord formation. Shimada M, Satoh N, Yokosawa H. Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. The Rel/NF-kappaB family is known to be involved in a wide variety of biological processes, including morphogenesis. In the present study, two protochordate cDNA clones encoding Rel/NF-kappaB proteins, named As-rel1 and As-rel2, were isolated from a fertilized egg cDNA library of the ascidian Halocynthia roretzi. The As-rel1 protein is a typical Rel/NF-kappaB family member, containing a Rel homology domain, a nuclear localization sequence and a C-terminal putative transcription activation domain, while the As-rel2 protein is a novel Rel/NF-kappaB family member that lacks a nuclear localization sequence and the C-terminal domain. Northern blot analyses showed that both transcripts were maternally expressed and that their expression changed during development of H. roretzi embryos. Although injection of the As-rel2 mRNA into H. roretzi fertilized eggs had little effect on embryonic development, injection of the As-rel1 mRNA interfered greatly with notochord formation, resulting in a shortened tail with a reduced number of notochord cells. In contrast, embryos co-injected with As-rel1 and As-rel2 mRNA developed normally, indicating that the As-rel2 protein rescued the defect in notochord formation induced by the injection of As-rel1 mRNA alone. These results strongly suggest that the As-rel1 protein functions as a suppressor in ascidian notochord formation, while the As-rel2 protein has an antagonistic effect on the action of the As-rel1 protein. PMID: 11284964 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 131: Nat Cell Biol. 2001 Apr;3(4):409-16. Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gelinas C, Fuchs EJ, Bedi A. Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, Maryland 21231, USA. TRAIL (tumour-necrosis factor-related apoptosis ligand or Apo2L) triggers apoptosis through engagement of the death receptors TRAIL-R1 (also known as DR4) and TRAIL-R2 (DR5). Here we show that the c-Rel subunit of the transcription factor NF-kappaB induces expression of TRAIL-R1 and TRAIL-R2; conversely, a transdominant mutant of the inhibitory protein IkappaBalpha or a transactivation-deficient mutant of c-Rel reduces expression of either death receptor. Whereas NF-kappaB promotes death receptor expression, cytokine-mediated activation of the RelA subunit of NF-kappaB also increases expression of the apoptosis inhibitor, Bcl-xL, and protects cells from TRAIL. Inhibition of NF-kappaB by blocking activation of the IkappaB kinase complex reduces Bcl-x L expression and sensitizes tumour cells to TRAIL-induced apoptosis. The ability to induce death receptors or Bcl-xL may explain the dual roles of NF-kappaB as a mediator or inhibitor of cell death during immune and stress responses. PMID: 11283615 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 132: J Neuroimmunol. 2001 Apr 2;115(1-2):199-202. Decreased expression of c-myc family genes in thymuses from myasthenia gravis patients. Nagata T, Onodera H, Ohuchi M, Suzuki Y, Tago H, Fujihara K, Ishii N, Sugamura K, Shoji Y, Handa M, Tabayashi K, Itoyama Y. Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Sendai 980-8574, Aoba, Japan. The thymus is a critical organ for the elimination of autoreactive T cells by apoptosis. We studied the expression of apoptosis-associated genes, bcl-xL, bad, caspase-3, and c-myc family genes in myasthenia gravis (MG) thymuses. We observed that the mRNA levels of myc family genes, c-myc and max, were markedly reduced in MG thymuses. These results indicate that c-myc-mediated signaling is abnormal in MG thymuses. The levels of molecules whose expressions are associated with myc, such as STAM, prothymosin-alpha, and NFkappaB, were also analyzed. Publication Types: Clinical Trial PMID: 11282171 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 133: J Bone Miner Res. 2001 Mar;16(3):501-10. Down-regulation of procollagen alpha1[I]] messenger RNA by titanium particles correlates with nuclear factor kappaB (NF-kappaB) activation and increased rel A and NF-kappaB1 binding to the collagen promoter. Roebuck KA, Vermes C, Carpenter LR, Fritz EA, Narayanan R, Glant TT. Department of Immunology/Microbiology, Rush University and Rush-Presbyterian-St Luke's Medical Center, Chicago, Illinois, USA. Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor kappaB (NF-kappaB), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen alpha1[I]. In this study, we identify three NF-kappaB binding sites within the human procollagen alpha1[I] gene promoter, show that titanium particles stimulate their binding of the NF-kappaB subunits Rel A (p65) and NF-kappaB1 (p50), and find NF-kappaB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-kappaB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-kappaB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-kappaB, suggesting the involvement of redox signals in NF-kappaB-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen alpha1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen alpha1[I] mRNA. Collectively, these results show that titanium particles can activate NF-kappaB signaling in osteoblasts and suggest that NF-kappaB binding to the collagen gene promoter has a functional role in the down-regulation of procollagen alpha1[I] gene transcription. PMID: 11277268 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 134: Nat Struct Biol. 2001 Apr;8(4):371-8. The leukemia-associated AML1 (Runx1)--CBF beta complex functions as a DNA-induced molecular clamp. Bravo J, Li Z, Speck NA, Warren AJ. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. We have determined the structure, at 2.6 A resolution, of the AML1 (Runx1) Runt domain--CBF beta--DNA ternary complex, the most common target for mutations in human leukemia. The structure reveals that the Runt domain DNA binding mechanism is unique within the p53 family of transcription factors. The extended C-terminal 'tail' and 'wing' elements adopt a specific DNA-bound conformation that clamps the phosphate backbone between the major and minor grooves of the distorted B-form DNA recognition site. Furthermore, the extended 'tail' mediates most of the NF-kappa B/Rel-like base-specific contacts in the major groove. The structure clearly explains the molecular basis for the loss of DNA binding function of the Runt domain--CBF beta complex as a consequence of the human disease-associated mutations in leukemogenesis and cleidocranial dysplasia. PMID: 11276260 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 135: J Mol Biol. 2001 Mar 30;307(3):785-98. Charging levels of four tRNA species in Escherichia coli Rel(+) and Rel(-) strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy. Sorensen MA. Department of Molecular Cell Biology, University of Copenhagen, Denmark. mas@biobase.dk Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation. One consequence of this deficiency is a tenfold increase in misincorporation at starved codons compared to the wild-type. Previous work had shown that the charging levels of tRNAs were the same in Rel(+) and Rel(-) strains and reduced, at most, two- to fivefold in both strains during starvation. The present reinvestigation of the charging levels of tRNA(2)(Arg), tRNA(1)(Thr), tRNA(1)(Leu) and tRNA(His) during starvation of isogenic Rel(+) and Rel(-) strains showed that starvation reduced charging levels tenfold to 40-fold. This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier. The determination of the charging levels of tRNA(2)(Arg) and tRNA(1)(Thr) during starvation were accurate enough to demonstrate that charging levels were at least fivefold lower in the Rel(-) strain compared to the Rel(+) strain. Together with other data from the literature, these new data suggest a simple model in which mis-incorporation increases as the substrate availability decreases and that ppGpp has no direct effect on enhancing translational accuracy at the ribosome. Copyright 2001 Academic Press. PMID: 11273701 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 136: Biochem J. 2001 Apr 1;355(Pt 1):29-38. Alpha-melanocyte-related tripeptide, Lys-d-Pro-Val, ameliorates endotoxin-induced nuclear factor kappaB translocation and activation: evidence for involvement of an interleukin-1beta193-195 receptor antagonism in the alveolar epithelium. Haddad JJ, Lauterbach R, Saade NE, Safieh-Garabedian B, Land SC. Oxygen Signalling Group, Centre for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K. j.j.haddad@dundee.ac.uk The potential anti-inflammatory role of alpha-melanocyte-stimulating hormone (alpha-MSH)-related tripeptide, lysine(11)-D-proline-valine(13) (KDPV), an analogue of interleukin (IL)-1beta(193-195) and an antagonist of IL-1beta/prostaglandin E(2), is not well characterized in the alveolar epithelium. In a model of foetal alveolar type II epithelial cells in vitro, we showed that lipopolysaccharide endotoxin (LPS) differentially, but selectively, induced the nuclear subunit composition of nuclear factor kappaB(1) (NF-kappaB(1)) (p50), RelA (p65) and c-Rel (p75), in parallel to up-regulating the DNA-binding activity (supershift indicating the presence of the p50-p65 complex). LPS accelerated the degradation of inhibitory kappaB-alpha (IkappaB-alpha), accompanied by enhancing its phosphorylation in the cytosolic compartment but not in the nucleus. KDPV suppressed, in a dose-dependent manner, the nuclear localization of p50, p65 and p75, an effect that led to the subsequent inhibition of NF-kappaB activation. Interleukin-1 receptor antagonist (IL-1ra) decreased the nuclear abundance of p50, p65 and p75, and subsequently depressed the DNA-binding activity induced by LPS. Analysis of the mechanism involved in the KDPV- and IL-1ra-mediated inhibition of NF-kappaB nuclear localization revealed a reversal in IkappaB-alpha phosphorylation and degradation, followed by cytosolic accumulation. LPS induced endogenous IL-1beta biosynthesis in a time-dependent manner; the administration of exogenous recombinant human interleukin 1 (rhIL-1) resulted in a dose-dependent activation of NF-kappaB. KDPV and IL-1ra abrogated the effect of rhIL-1. Pretreatment with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, an inhibitor of cyclo-oxygenase, blocked the LPS-induced activation of NF-kappaB. These results indicate the involvement of prostanoid-dependent (NSAID-sensitive) and IL-1-dependent (IL-1ra-sensitive) mechanisms mediating LPS-induced NF-kappaB translocation and activation, a pathway that is regulated, in part, by a negative feedback mechanism transduced through IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB. PMID: 11256945 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 137: Circulation. 2001 Jan 16;103(2):213-9. PPARalpha activators inhibit tissue factor expression and activity in human monocytes. Marx N, Mackman N, Schonbeck U, Yilmaz N, Hombach V, Libby P, Plutzky J. Department of Internal Medicine II-Cardiology, University of Ulm, Germany. nikolaus.marx@medizin.uni-ulm.de BACKGROUND: Tissue factor (TF), expressed on the surface of monocytes and macrophages in human atherosclerotic lesions, acts as the major procoagulant initiating thrombus formation in acute coronary syndromes. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a nuclear receptor family member, regulates gene expression in response to certain fatty acids and fibric acid derivatives. Given that some of these substances reduce TF activity in patients, we tested whether PPARalpha activators limit TF responses in human monocytic cells. METHODS AND RESULTS: Pretreatment of freshly isolated human monocytes or monocyte-derived macrophages with PPARalpha activators WY14643 and eicosatetraynoic acid (ETYA) led to reduced lipopolysaccharide (LPS)-induced TF activity in a concentration-dependent manner (maximal reduction to 43+/-8% with 250 micromol/L WY14643 [P:<0.05, n=5] and to 42+/-12% with 30 micromol/L ETYA [P:>0.05, n=3]). Two different PPARgamma activators (15-deoxy(_Delta12,14)-prostaglandin J(2) and BRL49653) lacked similar effects. WY14643 also decreased tumor necrosis factor-alpha protein expression in supernatants of LPS-stimulated human monocytes. Pretreatment of monocytes with WY14643 inhibited LPS-induced TF protein and mRNA expression without altering mRNA half-life. Transient transfection assays of a human TF promoter construct in THP-1 cells revealed WY14643 inhibition of LPS-induced promoter activity, which appeared to be mediated through the inhibition of nuclear factor-kappaB but not to be due to reduced nuclear factor-kappaB binding. CONCLUSIONS: PPARalpha activators can reduce TF expression and activity in human monocytes/macrophages and thus potentially reduce the thrombogenicity of atherosclerotic lesions. These data provide new insight into how PPARalpha-activating fibric acid derivatives and certain fatty acids might influence atherothrombosis in patients with vascular disease. PMID: 11208679 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 138: J Neurochem. 2001 Feb;76(4):1188-98. Erratum in: J Neurochem 2001 Apr;77(1):351. NF-kappaB is involved in the survival of cerebellar granule neurons: association of IkappaBbeta [correction of Ikappabeta] phosphorylation with cell survival. Koulich E, Nguyen T, Johnson K, Giardina C, D'mello S. Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, USA. The NF-kappaB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c-Rel. NF-kappaB activity is tightly controlled by IkappaB proteins which bind to NF-kappaB preventing its translocation to the nucleus. Activation of NF-kappaB is most often mediated by IkappaB degradation, which permits NF-kappaB to enter the nucleus. We investigated the role of NF-kappaB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF-kappaB activity: SN-50, N-tosyl-L-phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF-kappaB is required for neuronal survival. Gel-shift assays reveal three complexes that bind to the NF-kappaB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium-induced apoptosis, whereas overexpression of IkappaBalpha promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IkappaBalpha and IkappaBbeta is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c-Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IkappaBbeta is, however, reduced by low potassium treatment. Besides being necessary for high potassium-mediated neuronal survival, NF-kappaB is also involved in the survival-promoting effects of IGF-1 and cAMP as judged by the ability of SN-50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium-induced alterations in the DNA-binding activity of NF-kappaB. Taken together, our results show that NF-kappaB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF-kappaB-mediated survival of cerebellar granule neurons. PMID: 11181838 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 139: Cancer Lett. 2001 Mar 26;164(2):119-26. Capsaicin suppresses phorbol ester-induced activation of NF-kappaB/Rel and AP-1 transcription factors in mouse epidermis. Han SS, Keum YS, Seo HJ, Chun KS, Lee SS, Surh YJ. College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-gu, 151-742, Seoul, South Korea. Capsaicin, the principal pungent ingredient of hot chili peppers, has anti-inflammatory and analgesic properties and is currently used as a topical cream for the management of various neuropathic conditions. In the present study, topical application of capsaicin onto dorsal skin of female ICR mice strongly suppressed phorbol ester-stimulated activation of NF-kappaB via blockade of IkappaB-alpha degradation with subsequent inhibition of nuclear translocation of the functionally active NF-kappaB subunit, p65. Likewise, phorbol ester-induced activation of activator protein-1 (AP-1) was abolished by capsaicin pretreatment. Since altered transactivation of NF-kappaB and AP-1 has been implicated for neoplastic transformation and progression, the suppression of these transcription factors by capsaicin may account for its previously reported chemopreventive effects on mouse skin tumorigenesis as well as inflammation. PMID: 11179825 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 140: J Invest Dermatol. 2001 Jan;116(1):124-30. Dimethylfumarate is an inhibitor of cytokine-induced nuclear translocation of NF-kappa B1, but not RelA in normal human dermal fibroblast cells. Vandermeeren M, Janssens S, Wouters H, Borghmans I, Borgers M, Beyaert R, Geysen J. Janssen Research Foundation, Beerse, Belgium. mvanderm@janbe.jnj.com We previously demonstrated that the oral antipsoriatic dimethylfumarate is an inhibitor of cytokine-induced adhesion molecule expression in endothelial HUVEC cells. We now report the inhibitory effect of dimethylfumarate on tumor-necrosis-factor-alpha- or interleukin-1 alpha-induced intercellular adhesion molecule 1 expression in normal human dermal fibroblasts. Western blots of normal human dermal fibroblast cytoplasmic extracts showed that dimethylfumarate has minor effects on the I kappa B alpha, beta and epsilon proteins: their cytokine-induced degradation and resynthesis is only slowed down, an effect most prominently observed for I kappa B beta. No inhibitory effect of dimethylfumarate was observed on cytokine-induced RelA/p65 or c-Rel accumulation in nuclear extracts of cytokine-treated normal human dermal fibroblast cells. In contrast, cytokine-induced nuclear factor kappa B1/p50 nuclear accumulation was specifically inhibited by dimethylfumarate. This inhibitory effect on nuclear factor kappa B1 nuclear localization in normal human dermal fibroblasts proved sufficient to inhibit nuclear factor kappa B1-RelA binding to nuclear factor kappa B consensus oligonucleotides in DNA binding assays. Likewise, cytokine-induced activation of a pNF kappa B::luciferase reporter construct in transiently transfected normal human dermal fibroblasts was inhibited by dimethylfumarate. The observations support a mechanistic model for the oral antipsoriatic dimethylfumarate in which lowering of nuclear factor kappa B1 leads to changes in the nuclear factor kappa B1-RelA nuclear balance and inhibition of cytokine-induced adhesion molecule expression in normal human dermal fibroblasts. PMID: 11168807 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 141: Mol Pharmacol. 2001 Feb;59(2):302-9. The role of NF-kappaB as a survival factor in environmental chemical-induced pre-B cell apoptosis. Mann KK, Doerre S, Schlezinger JJ, Sherr DH, Quadri S. Boston University Schools of Public Health and Medicine, Department of Environmental Health, Boston, Massachusetts 02118, USA. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental chemicals that suppress the immune system at multiple levels, including at the level of B cell development in the bone marrow microenvironment. Specifically, PAH induce preB cell apoptosis in primary bone marrow cultures and in cocultures of an early preB cell line (BU-11) and a bone marrow stromal cell line (BMS2). Previous studies focused on the molecular mechanisms through which PAH induce stromal cells to deliver an apoptosis signal to adjacent preB cells. Apoptosis signaling within the preB cell itself was not investigated. Here, the role of NF-kappaB, a lymphocyte survival factor, in PAH-induced preB cell apoptosis was assessed. Analysis of DNA-binding proteins extracted from the nuclei of untreated BU-11 cells indicated DNA-binding complexes comprising NF-kappaB subunits p50, c-Rel, and/or Rel A. NF-kappaB down-regulation with previously described inhibitors induced BU-11 cell apoptosis, demonstrating that the default apoptosis pathway blocked by NF-kappaB is functional at this early stage in B cell development. Similarly, exposure of BU-11/BMS2 cocultures to 7,12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH, down-regulated nuclear Rel A and c-Rel before overt apoptosis. Finally, ectopic expression of Rel A or c-Rel rescued BU-11 cells from DMBA-induced apoptosis. These results extend previous observations by demonstrating that 1) NF-kappaB is a survival factor at an earlier stage of B cell development than previously appreciated and 2) NF-kappaB down-regulation is likely to be part of the molecular mechanism resulting in PAH-induced preB cell apoptosis. These results suggest nonclonally restricted, PAH-mediated suppression of B lymphopoiesis. PMID: 11160867 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 142: Hum Pathol. 2000 Dec;31(12):1482-90. Constitutive expression of NF-kappa B is a characteristic feature of mycosis fungoides: implications for apoptosis resistance and pathogenesis. Izban KF, Ergin M, Qin JZ, Martinez RL, Pooley RJ JR, Saeed S, Alkan S. Department of Pathology, Loyola University Medical Center, Maywood, IL 60153, USA. The NF-kappa B family of transcription factors is an important regulator of genes expressed during inflammatory responses, immunoglobulin (Ig) class switching, cellular differentiation, and apoptosis. Recently, members of the NF-kappaB family, including p65(Rel A), have been implicated in promoting survival of various hematopoeitic neoplasms, including T cell malignancies such as adult T cell leukemia-lymphoma. We investigated the expression of active NF-kappa B p65(Rel A) in cases of mycosis fungoides (MF) and the effect of chemical inhibitors of NF-kappa B on apoptosis in cutaneous T cell lymphoma (CTCL) cell lines. Paraffin-embedded tissues from 23 cutaneous lesions and a single lymph node biopsy from patients diagnosed with MF were evaluated for p65(Rel A) expression by using a monoclonal mouse antibody that detects the activated form of p65(Rel A). Apoptosis after treatment with the NF-kappa B inhibitors gliotoxin, MG132, BAY 11-7082, and BAY 11-7085 was quantitatively measured in the CTCL cell lines HuT-78 and HH by propidium iodide (PI)/cell cycle analysis for detection of a hypodiploid (sub-G(0)) population and by determination of increased Annexin V/7-amino-actinomycin D (7-AAD) expression. Nuclear extracts from CTCL cells before and after chemical inhibition were analyzed for NF-kappa B nuclear DNA-binding activity by electrophoretic mobility shift assay (EMSA) with quantitative densitometry. Nuclear expression of p65(Rel A) before and after treatment with the various inhibitory compounds was measured by immunofluorescence staining in each CTCL cell line. Neoplastic T lymphocytes from 22 of 24 cases of MF showed strong nuclear and cytoplasmic expression of active p65(Rel A). Compared with untreated control cells, a marked increase in apoptosis, a significant decrease in NF-kappa B DNA-binding activity, and a marked decrease in nuclear p65(Rel A) expression were seen in cells from both CTCL cell lines after chemical NF-kappa B inhibition. These data show that the active form of NF-kappa B p65(Rel A) is commonly expressed in neoplastic T lymphocytes in patients with MF. In CTCL cell lines, the significant decrease in nuclear NF-kappa B expression and the marked increase in spontaneous apoptosis caused by chemical NF-kappa B inhibition suggest a critical role for NF-kappa B in the pathogenesis and tumor cell maintenance of CTCLs. HUM PATHOL 31:1482-1490. Copyright 2000 by W.B. Saunders Company PMID: 11150373 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 143: Cancer. 2000 Dec 1;89(11):2274-81. Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma. Tai DI, Tsai SL, Chang YH, Huang SN, Chen TC, Chang KS, Liaw YF. Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan, Republic of China. BACKGROUND: Nuclear factor kappaB (NF-kappaB) is a transcription factor that plays important roles in cell proliferation and in immunity against viral infections. NF-kappaB is a dimer of Rel proteins that is sequestered in the cytoplasm as an inactive form through interaction with an inhibitory kappaB (IkappaB) protein. When IkappaB is degraded, the NF-kappaB dimer will enter the nucleus to activate the target genes. Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection may activate NF-kappaB and, thus, may modulate cell apoptosis and may be associated with oncogenesis. The role of NF-kappaB in hepatocellular carcinoma (HCC) has not yet been explored. METHODS: Immunohistochemical staining to search for active nuclear RelA and nuclear IkappaBalpha proteins were done on formalin fixed liver tissues from 65 patients with HCC and from 9 normal control participants. Nuclear extracts of fresh-frozen tumor and nontumor liver tissues from 37 patients with HCC and from 7 normal controls were tested for NF-kappaB-DNA binding activity by electrophoretic mobility shift assay. The RelA and IkappaBalpha protein expressions were studied by Western blot analysis. RESULTS: Nuclear NF-kappaB stainings were significantly more abundant in HBV-infected or HCV-infected tumors as well as nontumor parts of HCC compared with normal controls. Nuclear NF-kappaB DNA binding activity and nuclear RelA protein expression were greater in tumor tissue compared with nontumor tissue, whereas cytosolic IkappaBalphs protein expression was generally greater in nontumor tissue compared with tumor tissue. CONCLUSIONS: Constitutive activation of NF-kappaB was found more frequently in tumor tissue compared with nontumor tissue. It is possible that NF-kappaB overexpression accompanied by dysregulation of IkappaBalpha may play a role in the hepatocarcinogenesis of HBV or HCV infection. PMID: 11147598 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 144: Br J Haematol. 2000 Nov;111(2):618-25. The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma. Goff LK, Neat MJ, Crawley CR, Jones L, Jones E, Lister TA, Gupta RK. ICRF Medical Oncology Unit, Department of Medical Oncology, St. Bartholomew's Hospital, London, UK. goffl@icrf.icnet.uk Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B-cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13-16. Using real-time quantitative polymerase chain reaction (RQ-PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, beta2-microglobulin and CD8alpha, that lie close to REL on 2p. There was no correlation apparent between 2p13-16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real-time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas. PMID: 11122110 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 145: J Virol. 2001 Jan;75(1):215-25. Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein. Su F, Theodosis CN, Schneider RJ. Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA. Chronic infection with hepatitis B virus (HBV) promotes a high level of liver disease and cancer in humans. The HBV HBx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor necrosis factor alpha (TNF-alpha) and etopocide. Paradoxically, HBx activates members of the NF-kappaB transcription factor family, some of which are antiapoptotic in function. HBx induces expression of Myc protein family members in certain settings, and Myc can sensitize cells to killing by TNF-alpha. We therefore examined the roles of NF-kappaB, c-Myc, and TNF-alpha in apoptotic killing of cells by HBx. RelA/NF-kappaB is shown to be induced by HBx and to suppress HBx-mediated apoptosis. HBx also induces c-Rel/NF-kappaB, which can promote apoptotic cell death in some contexts or block it in others. Induction of c-Rel by HBx was found to inhibit its ability to directly mediate apoptotic killing of cells. Thus, HBx induction of NF-kappaB family members masks its ability to directly mediate apoptosis, whereas ablation of NF-kappaB reveals it. Investigation of the role of Myc protein demonstrates that overexpression of Myc is essential for acute sensitization of cells to killing by HBx plus TNF-alpha. This study therefore defines a specific set of parameters which must be met for HBx to possibly contribute to HBV pathogenesis. PMID: 11119591 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 146: Oncogene. 2000 Nov 16;19(48):5498-506. The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells. Kim DW, Gazourian L, Quadri SA, Romieu-Mourez R, Sherr DH, Sonenshein GE. Department of Biochemistry, Women's Health, Boston University School of Medicine, Massachusetts 02118, USA. NF-kappaB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-kappaB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-kappaB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells. PMID: 11114727 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 147: EMBO J. 2000 Dec 1;19(23):6351-60. The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression. Grossmann M, O'Reilly LA, Gugasyan R, Strasser A, Adams JM, Gerondakis S. The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia. Rel and RelA, individually dispensable for lymphopoiesis, serve unique functions in activated B and T cells. Here their combined roles in lymphocyte development were examined in chimeric mice repopulated with c-rel(-/-) rela(-/-) fetal liver hemopoietic stem cells. Mice engrafted with double-mutant cells lacked mature IgM(lo)IgD(hi) B cells, and numbers of peripheral CD4(+) and CD8(+) T cells were markedly reduced. The absence of mature B cells was associated with impaired survival that coincided with reduced expression of bcl-2 and A1. bcl-2 transgene expression not only prevented apoptosis and increased peripheral B-cell numbers, but also induced further maturation to an IgM(lo)IgD(hi) phenotype. In contrast, the survival of double-mutant T cells was normal and the bcl-2 transgene could not rectify the peripheral T-cell deficit. These findings indicate that Rel and RelA serve essential, albeit redundant, functions during the later antigen-independent stages of B- and T-cell maturation, with these transcription factors promoting the survival of peripheral B cells in part by upregulating Bcl-2. PMID: 11101508 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 148: Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12705-10. Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages. Sanjabi S, Hoffmann A, Liou HC, Baltimore D, Smale ST. Howard Hughes Medical Institute and Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095-1662, USA. A major challenge in the study of gene regulation by NF-kappaB/Rel transcription factors is to understand, at the biological and mechanistic levels, the selective functions of individual Rel family members. To study selectivity, we have examined the NF-kappaB/Rel protein binding site (Rel site) within the IL-12 p40 promoter. IL-12 is a proinflammatory cytokine expressed by activated macrophages that serves as an essential inducer of T helper 1 cell development. In nuclear extracts from lipopolysaccharideactivated macrophages, the predominant Rel dimers capable of binding the IL-12 p40 Rel site were the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The two heterodimers bound the site with comparable affinities and exhibited comparable transactivation activities. In striking contrast, p40 mRNA and protein concentrations were reduced dramatically in c-Rel(-/-) macrophages and only modestly in p65(-/-) macrophages. Other proinflammatory cytokine mRNAs and proteins were not significantly reduced in c-Rel(-/-) macrophages. These results reveal that a c-Rel-containing complex is an essential and selective activator of p40 transcription, which may reflect unique regulatory mechanisms or biological functions of IL-12. Furthermore, because selectivity was not observed in vitro or in transient transactivation experiments, these findings suggest that an understanding of the selectivity mechanism may require an analysis of the endogenous p40 locus. PMID: 11058167 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 149: Immunol Rev. 2000 Aug;176:134-40. Rel/NF-kappaB transcription factors: key mediators of B-cell activation. Gugasyan R, Grumont R, Grossmann M, Nakamura Y, Pohl T, Nesic D, Gerondakis S. The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Victoria, Australia. The Rel/nuclear factor (NF)-kappaB family of transcription factors have been implicated in the regulation of a wide variety of genes, in particular those encoding proteins crucial to the function of the immune system. Through the use of mutant mice that lack one or more of these proteins, we have begun to examine the individual and combined roles of Rel, RelA and NF-kappaB1 in B-cell development and function. Here we outline and discuss how these transcription factors operate as differentiation stage-specific regulators of B-cell development, survival, division and immunoglobulin expression, emphasizing those Rel/NF-kappaB-regulated genes that mediate these functions. Publication Types: Review Review, Tutorial PMID: 11043773 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 150: J Immunol. 2000 Oct 15;165(8):4592-7. The Rel family member P50 mediates cytokine-induced C-reactive protein expression by a novel mechanism. Cha-Molstad H, Agrawal A, Zhang D, Samols D, Kushner I. Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA. Transcription of C-reactive protein (CRP) in Hep 3B cells is induced by IL-6, acting through C/EBP isoforms and STAT3. IL-1beta, which alone has no effect, greatly enhances IL-6-induced transcription by unknown mechanisms. Because IL-1beta activates the NF-kappaB system, we explored the effects of overexpressed Rel family members on CRP expression. Unexpectedly, transactivation assays in transiently transfected Hep 3B cells showed p50 overexpression to markedly induce CRP transcription, acting in a region 3' to -86. In the presence of overexpressed p50, IL-1beta induced a 3-fold increase in CRP expression, and responses to IL-6 and to IL-6 plus IL-1beta were 4-fold greater than seen in cells without p50 overexpression. In contrast, overexpressed p65 abolished CRP induction by p50 and by cytokines. EMSA studies demonstrated that recombinant p50 bound to a nonconsensus kappaB site overlapping the proximal C/EBP binding site on the CRP promoter. Mutation of a polypyrimidine tract in the p50-binding site inhibited the transactivating effect of cytokines. P50- but not p65-containing dimers were found in nuclei of Hep 3B cells 18 h after stimulation with IL-1beta, when C/EBPbeta is greatly activated, in the presence or absence of IL-6. These findings suggest that IL-1beta induces nuclear translocation of p50-containing dimers and that p50 interacts with C/EBPbeta activated by both IL-6 and IL-1beta to induce CRP expression. PMID: 11035101 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 151: J Exp Med. 2000 Oct 16;192(8):1175-82. Nuclear factor kappa B is required for the development of marginal zone B lymphocytes. Cariappa A, Liou HC, Horwitz BH, Pillai S. Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts 02129, USA. Although immunoglobulin (Ig)M(hi)IgD(lo/-)CD21(hi) marginal zone B cells represent a significant proportion of naive peripheral splenic B lymphocytes, few of the genes that regulate their development have been identified. This subset of peripheral B cells fails to emerge in mice that lack nuclear factor (NF)-kappa Bp50. Less drastic reductions in marginal zone B cell numbers are also seen in the spleens of recombination activating gene (Rag)-2(-/-) mice reconstituted with NF-kappa Bp65(-/-) fetal liver cells and in c-Rel(-/-) mice. In contrast, steady-state levels of IgD(hi) splenic follicular B cells are not significantly reduced in the absence of NF-kappa Bp50, NF-kappa Bp65, or c-Rel. Reconstitution of B cells in Rag-2(-/-) mice with a mixture of p50(-/-)/p65(-/-) fetal liver cells and Rag-2(-/-) bone marrow cells revealed that the generation of marginal zone B cells requires the expression of NF-kappa B in developing B cells, as opposed to supporting cells. PMID: 11034607 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 152: FEMS Microbiol Lett. 2000 Sep 15;190(2):195-201. Characterization of the relA/spoT gene from Bacillus stearothermophilus. Wendrich TM, Beckering CL, Marahiel MA. Department of Chemistry, Philipps-University, Marburg, Germany. By use of degenerate primers, we amplified a fragment of a relA/spoT homologous gene from Bacillus stearothermophilus. Chromosomal walking enabled us to sequence the entire gene and its flanking regions. The primary sequence of the gene product is 78% identical to the RelA/SpoT homologue of Bacillus subtilis and both gene loci share a similar genetic organization. The B. stearothermophilus rel gene was analyzed in vivo by heterologous expression in the B. subtilis relA deletion strain TW30, and is shown to complement the growth defects of TW30. The recombinant RelBst protein was detected by Western immunoanalysis, and synthesizes guanosine-3'-diphosphate-5'-(tri)diphosphate ((p)ppGpp) after amino acid stress and carbon starvation. These in vivo data, the genetic organization, and the primary structure compared to other RelA/SpoT homologues provide circumstantial evidence that the identified gene encodes the only (p)ppGpp synthetase in B. stearothermophilus presumed to serve also as (p)ppGpp hydrolase. PMID: 11034279 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 153: Mech Dev. 2000 Oct;97(1-2):149-55. Dynamic expression and activity of NF-kappaB during post-natal mammary gland morphogenesis. Brantley DM, Yull FE, Muraoka RS, Hicks DJ, Cook CM, Kerr LD. Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrated that DNA-binding complexes containing p50 and RelA were abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice was highest during pregnancy, decreased to near undetectable levels during lactation, and was elevated during involution. This highly regulated pattern of activity was consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha. PMID: 11025216 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 154: Microbes Infect. 2000 Sep;2(11):1311-20. Characterisation of NF-kappa B complexes in Theileria parva-transformedT cells. Machado J Jr, Fernandez PC, Baumann I, Dobbelaere DA. Laboratory of Molecular Pathology, Institute of Animal Pathology, University of Berne, Langgass-Strasse 122, 3012, Berne, Switzerland. Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells. PMID: 11018447 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 155: Eur J Immunol. 2000 Jul;30(7):1967-76. CpG ODN-mediated regulation of IL-12 p40 transcription. Takeshita F, Klinman DM. Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda 20892, USA. Oligodeoxynucleotides (ODN) containing CpG motifs activate RAW 264.7 mouse macrophages and RPMI 8226 human myeloma cells to produce IL-12 p40. Using deletion and site-directed mutagenesis, the nuclear factor (NF)-kappaB half-site and the CCAAT/enhancer binding protein (C/EBP) recognition site were identified as potent cis-acting elements in CpG ODN-mediated IL-12 p40 promoter activation. Several NF-kappaB/Rel proteins competed for binding to the NF-kappaB half-site. The p65/c-Rel and p65/p50 heterodimer occupied this site shortly after CpG ODN administration (0.5-2 h), while the p50/c-Rel heterodimer dominated binding in the late stage (8-12 h). The induction of p50/c-Rel heterodimer was associated with a significant expression of IL-12 p40 mRNA. C/EBPbeta also contributed to CpG ODN-mediated IL-12 p40 promoter activation. PMID: 10940886 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 156: J Bacteriol. 2000 Sep;182(17):4889-98. The stringent response of Mycobacterium tuberculosis is required for long-term survival. Primm TP, Andersen SJ, Mizrahi V, Avarbock D, Rubin H, Barry CE 3rd. Tuberculosis Research Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20852, USA. The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions. PMID: 10940033 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 157: J Biol Chem. 2000 Oct 20;275(42):32592-7. Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. Wang D, Westerheide SD, Hanson JL, Baldwin AS Jr. Department of Biology, Curriculum in Genetics and Molecular Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA. Nuclear factor kappaB (NF-kappaB)/Rel transcription factors are key regulators of a variety of genes involved in immune and inflammatory responses, growth, differentiation, apoptosis, and development. In unstimulated cells, NF-kappaB/Rel proteins are sequestered in the cytoplasm by IkappaB inhibitor proteins. Many extracellular stimuli, such as tumor necrosis factor alpha (TNFalpha), cause rapid phosphorylation of IkappaB at N-terminal serine residues leading to ubiquitination and degradation of the inhibitor. Subsequently, NF-kappaB proteins translocate to the nucleus and activate gene expression through kappaB response elements. TNFalpha, as well as certain other stimuli, also induces the phosphorylation of the NF-kappaB proteins. Previously, we have shown that TNFalpha induces RelA/p65 phosphorylation at serine 529 and that this inducible phosphorylation increases NF-kappaB transcriptional activity on an exogenously supplied reporter (). In this report, we demonstrate that casein kinase II (CKII) interacts with p65 in vivo and can phosphorylate p65 at serine 529 in vitro. A CKII inhibitor (PD144795) inhibited TNFalpha-induced p65 phosphorylation in vivo. Furthermore, our results indicate that the association between IkappaBalpha and p65 inhibits p65 phosphorylation by CKII and that degradation of IkappaBalpha allows CKII to phosphorylate p65 to increase NF-kappaB transactivation potential. These data may explain the ability of CKII to modulate cell growth and demonstrate a mechanism whereby CKII can function in an inducible manner. PMID: 10938077 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 158: Mol Endocrinol. 2000 Aug;14(8):1222-34. CBP (CREB binding protein) integrates NF-kappaB (nuclear factor-kappaB) and glucocorticoid receptor physical interactions and antagonism. McKay LI, Cidlowski JA. Molecular Endocrinology Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA. Nuclear factor-kappaB (NF-kappaB) and the glucocorticoid receptor (GR) are transcription factors with opposing actions in the modulation of immune/inflammatory responses. NF-kappaB induces the expression of proinflammatory genes, while GR suppresses immune function in part by suppressing expression of the same genes. Previously, we demonstrated that physiological antagonism between NF-kappaB and GR is due to a mutual transcriptional antagonism that requires the p65 subunit of NF-kappaB and multiple domains of GR (1). To elucidate the mechanism(s) of NF-kappaB p65 and GR transcriptional antagonism, we analyzed the interactions of wild-type p65 and p65 RHD (rel homology domain, a dominant negative mutant of p65 which lacks a transactivation domain) with GR. We show that p65RHD blocks p65-mediated transactivation, yet does not block the repression of GR transactivation by p65, indicating that transcriptional activity by p65 is not required to repress GR function. Both p65 and p65 RHD physically interact with GR, but only intact p65 represses GR-mediated signaling, implicating the p65 transactivation domain in the transcriptional repression of GR. To further characterize p65-GR interactions, we examined the role of the transcriptional co-integrator CREB binding protein (CBP) in their mutual antagonism. GR-mediated repression of p65 transactivation and p65-mediated repression of GR transactivation, as well as the physical interaction between NF-kappaB and GR, are enhanced by CBP. GR bound to the antagonist RU 486, although transcriptionally inactive, retains the ability to repress p65 transactivation. However, CBP does not physically interact with antagonist-bound GR and does not enhance its repressive effect on p65. These data suggest that CBP functions as an integrator of p65/GR physical interaction, rather than as a limiting cofactor for which p65 and GR compete. PMID: 10935546 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 159: J Biol Chem. 2000 Oct 13;275(41):32338-46. NF-kappa B activity is required for the deregulation of c-myc expression by the immunoglobulin heavy chain enhancer. Kanda K, Hu HM, Zhang L, Grandchamps J, Boxer LM. Center for Molecular Biology in Medicine, Veterans Affairs Palo Alto Health Care System and the Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA. The c-myc gene is translocated to one of the immunoglobulin genes in Burkitt's lymphoma resulting in deregulated expression of c-myc. Several enhancers have been shown to be important for expression of the immunoglobulin heavy chain gene. Four enhancer regions (murine-hypersensitive sites (MHS) 1, 2, 3, and 4) located 3' of the murine immunoglobulin heavy chain gene play a role in activating expression of the translocated c-myc gene. The enhancer regions also result in a shift in transcriptional initiation from the P2 promoter to P1 that is characteristic of the translocated c-myc allele. We found that the most 3' enhancer region (MHS4) activated the c-myc promoter by 46-fold in the Raji Burkitt's lymphoma cell line, and it was the most active enhancer in these cells. The addition of enhancer regions MHS1,2 and 3 to MHS4 increased c-myc transcription by an additional 3-fold and resulted in the full promoter shift from P2 to P1. By deletion analysis of enhancer region MHS4, we located a region that was critical for the transcriptional activity of MHS4. Electrophoretic mobility shift assay analysis revealed that NF-kappaB/Rel family members bound to this region. Mutation of the NF-kappaB binding site abolished both the enhancer activity and the promoter shift activity of MHS4. An active NF-kappaB site was also identified in the human HS4 enhancer. Inhibition of c-myc promoter activity driven by the immunoglobulin enhancers was observed with expression of a super-repressor IkappaBalpha construct. These results indicate that the NF-kappaB/Rel transcription factors play an important role in the deregulation of the translocated c-myc gene in Burkitt's lymphoma and suggest that interference with NF-kappaB function may represent a new approach to the treatment of Burkitt's lymphoma. PMID: 10931834 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 160: FASEB J. 2000 Aug;14(11):1471-84. Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers. Schulte R, Grassl GA, Preger S, Fessele S, Jacobi CA, Schaller M, Nelson PJ, Autenrieth IB. Max von Pettenkofer-Institut fur Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universitat Munchen, D-80336 Munchen, Germany. Enteropathogenic YERSINIA: bacteria trigger the production of the proinflammatory chemokine IL-8, an important chemokine for the recruitment of polymorphonuclear leukocytes (PMN). YERSINIA: is resistant to phagocytosis by PMN, and the recruitment of these cells is thought to be part of a pathogenic strategy of YERSINIA: to establish infection by allowing the pathogen to gain access to, and disseminate within, host tissue. We report here that YERSINIA: expressing the outer membrane protein invasin triggers IL-8 production in epithelial cells. The 195 carboxyl-terminal amino acids of invasin when linked to latex beads are sufficient to trigger IL-8 production. By means of IL-8 promoter reporter gene assays and electrophoretic mobility shift assay experiments, the minimal optimal region of the IL-8 promoter responsive to invasin was identified and invasin-responsive control elements were characterized. Invasin-induced activation of the IL-8 promoter was found to be mediated through a previously identified NF-kappaB element. This NF-kappaB binding site preferentially binds Rel p65-p65 homodimers as well as some p50-p65 heterodimers in response to stimulation by invasin. Invasin-induced NF-kappaB activation correlated with degradation of IkappaBalpha and the inhibition of NF-kappaB by specific inhibitors of IkappaB activation blocked invasin-induced IL-8 secretion. Invasin-triggered IL-8 production does not depend on invasin-triggered uptake of bacteria, and is independent of a functional PI3-kinase. This report is the first to demonstrate the molecular basis of IL-8 production triggered by enteropathogenic bacteria. Together, these data elucidate the possible early pathomechanisms operating in YERSINIA: infection and may have implications for the design of novel therapeutics directed against this enteropathogen. PMID: 10928981 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 161: Am J Physiol Lung Cell Mol Physiol. 2000 Aug;279(2):L302-11. Activation of NF-kappaB induced by H(2)O(2) and TNF-alpha and its effects on ICAM-1 expression in endothelial cells. True AL, Rahman A, Malik AB. Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA. Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappaB in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H(2)O(2) and TNF-alpha in mediating activation of NF-kappaB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H(2)O(2) added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H(2)O(2) also failed to induce NF-kappaB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappaB-driven gene expression approximately 10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H(2)O(2)- and TNF-alpha-induced NF-kappaB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappaB p65 in HMEC-1 cells, whereas H(2)O(2) had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappaB to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappaB p65 and confer transcriptional competency to NF-kappaB. PMID: 10926553 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 162: J Biol Chem. 2000 Jun 30;275(26):19661-6. Glucocorticoids induce proteasome C3 subunit expression in L6 muscle cells by opposing the suppression of its transcription by NF-kappa B. Du J, Mitch WE, Wang X, Price SR. Renal Division, Emory University, Atlanta, Georgia 30322, USA. Muscle wasting in catabolic conditions results from activation of the ubiquitin-proteasome proteolytic pathway by a process that requires glucocorticoids and is generally associated with increased levels of mRNAs encoding components of this proteolytic system. In L6 muscle cells, dexamethasone stimulates proteolysis and increases the amount of the proteasome C3 subunit protein by augmenting its transcription. Transfection studies with human C3 promoter-luciferase reporter genes and electrophoretic mobility shift assays revealed that a NF-kappaB.protein complex containing Rel A is abundant in L6 muscle cell nuclei. Glucocorticoids stimulate C3 subunit expression by antagonizing the interaction of this NF-kappaB protein with an NF-kappaB response element in the C3 subunit promoter region. Dexamethasone also increased the cytosolic amounts of the NF-kappaB p65 subunit and the IkappaBalpha inhibitor proteins in L6 cells. Incubation of L6 cells with a cytokine mixture not only increased the amount of activated NF-kappaB but also decreased C3 promoter activity and lowered endogenous C3 subunit mRNA. Thus, NF-kappaB is a repressor of C3 proteasome subunit transcription in muscle cells, and glucocorticoids stimulate C3 subunit expression by opposing this suppressor action. PMID: 10867022 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 163: Chem Biol Interact. 2000 May 1;126(2):137-57. Identification of NF-kappaB in the marine fish Stenotomus chrysops and examination of its activation by aryl hydrocarbon receptor agonists. Schlezinger JJ, Blickarz CE, Mann KK, Doerre S, Stegeman JJ. Biology Department, Redfield 342, MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor kappaB (NF-kappaB) that rapidly activates genes encoding cytokines, cell surface receptors, cell adhesion molecules and acute phase proteins. Evidence suggests that xenobiotic compounds also may alter the activation of NF-kappaB. This study had a dual objective of identifying members of the Rel family and examining their activation by xenobiotic compounds in a marine fish model, scup (Stenotomus chrysops). A DNA-protein crosslinking technique demonstrated that liver, kidney and heart each had at least three nuclear proteins that showed specific binding to an NF-kappaB consensus sequence, with molecular weights suggesting that the proteins potentially corresponded to mouse p50, p65 (RelA) and c-rel. In addition, an approximately 35kD NF-kappaB binding protein was evident in liver and kidney. The 50 kD protein was immunoprecipitated by mammalian p50-specific antibodies. The presence of Rel members in fish implied by those results was confirmed by RT-PCR cloning of a Rel homology domain (an apparent c-rel) from scup liver. NF-kappaB activation occurred in vehicle-treated fish, but this appeared to decrease over time. In fish treated with 0.01 or 1 mg 3,3',4,4', 5-pentachlorobiphenyl per kg, NF-kappaB activation in liver did not decrease, and there was a 6-8-fold increase in activation 16-18 days following treatment. Treatment with 10 mg benzo[a]pyrene/kg had no effect on NF-kappaB-DNA binding, either at 3 or 6 days following treatment. The data show that the Rel family of proteins is present in fish, represented at least by a p50/105 homologue, and support a hypothesis that some aryl hydrocarbon receptor agonists can activate NF-kappaB in vivo. PMID: 10862814 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 164: Blood. 2000 Jun 15;95(12):3804-8. Comment in: Blood. 2001 Mar 1;97(5):1518-21. Synergistic induction of HLA class I expression by RelA and CIITA. Girdlestone J. Anatomy Department, MRC Centre for Immune Regulation, The Medical School, University of Birmingham, Birmingham, UK. girdlesj@bham.ac.uk The major histocompatibility complex (MHC) class I genes are induced synergistically by interferons (IFN) and tumor necrosis factor (TNF), a response thought to involve the cooperative action of Rel/NF-kB and interferon regulatory factor (IRF) transcription factors. The IFN-gamma-inducible class II transcriptional activator (CIITA) has recently been shown to transactivate MHC class I as well as class II genes, and this investigation shows that CIITA synergizes strongly with RelA to stimulate HLA class I expression. The functional interaction of CIITA and RelA requires both promoter elements and the upstream Rel binding site and is not seen with a class II reporter. The promoter elements necessary for CIITA action are also required for induction by IFN-alpha. HLA-A and HLA-B loci respond differentially to IFNs, and we identify locus-specific differences in critical promoter elements in addition to known polymorphisms in the Rel and IRF binding sites. The HLA-A promoter is transactivated relatively poorly by CIITA and does not interact detectably with CREB proteins implicated in CIITA recruitment, but the synergism with RelA can compensate for this weakness. The present findings illustrate that multiple transcription factors cooperate to regulate class I expression and that their relative importance differs according to the locus and cell type examined. (Blood. 2000;95:3804-3808) PMID: 10845913 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 165: Neurosci Lett. 2000 Jun 16;287(1):17-20. Activation of nuclear factor-kB in the spinal cord of experimental allergic encephalomyelitis. Pahan K, Schmid M. Department of Oral Biology, University of Nebraska Medical Center, Lincoln, 40th and Holdrege, Nebraska, NE 68583, USA. kpahan@unmc.edu The DNA-binding activity of nuclear factor (NF-kB) was found to be induced in the spinal cord of rats with experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), from the onset of the disease. This activation of NF-kB persisted throughout the disease period and decreased thereafter in the recovery phase. Supershift analysis of NF-kB DNA-binding activity in nuclear extracts of spinal cords showed that RelA/p65 and p50 subunits but not c-Rel/p75, RelB/p68 and p52 subunits were involved in DNA binding. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB activation, markedly inhibited the in vivo activation of NF-kB in the spinal cord of EAE rats and attenuated the clinical symptoms of EAE. These studies suggest that activation of NF-kB plays an important role in the pathogenesis of EAE and inhibitors of NF-kB activation may have therapeutic importance in MS. PMID: 10841980 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 166: J Biol Chem. 2000 Aug 11;275(32):24383-91. Tumor necrosis factor-alpha activation of NF-kappa B requires the phosphorylation of Ser-471 in the transactivation domain of c-Rel. Martin AG, Fresno M. Centro de Biologia Molecular Severo Ochoa, Cantoblanco, Madrid 28049, Spain. Activation of the transcription factor NF-kappaB is controlled at two levels in resting T cells: an initial activation induced by the triggering of the TcR.CD3 complex and a second phase controlled by paracrine- or autocrine-secreted TNFalpha. The initial phase is regulated by p65 (RelA), whereas the second one is mainly dependent on c-Rel. We describe here a mutant clone, D6, derived from the parental T lymphoblastic line Jurkat that fails to activate NF-kappaB upon TNFalpha stimulation. This clone had no alteration in tumor necrosis factor-alpha (TNFalpha) signaling pathways nor in IkappaBalpha, -beta, or -epsilon expression and degradation. However, TNFalpha induced an exacerbated apoptotic response in this clone compared with Jurkat cells. This mutant clone showed a defect in the intermediate-late translocation of c-Rel to the nucleus promoted by TNFalpha stimulation, whereas early translocation is not affected. Activation or translocation of p65-containing complexes was not altered in this mutant clone. Sequencing of the c-Rel gene from this clone revealed a mutation of Ser-471 to Asn in the transactivation domain. The mutant S471N transactivation domain fused to the Gal4 DNA binding domain could not be activated by TNFalpha, unlike the wild type. Moreover, the overexpression of the mutant protein c-Rel S471N into Jurkat cells abolished TNFalpha-induced NF-kappaB activity, thus demonstrating that this mutation is responsible for the failure of TNFalpha stimulation of NF-kappaB. Moreover, extracts from TNFalpha-stimulated Jurkat cells phosphorylated in vitro recombinant wild type GST-c-Rel 464-481 but not the GST-c-Rel mutant. Thus, TNFalpha-induced phosphorylation of Ser-471 seems to be absolutely necessary for TNFalpha activation of c-Rel. PMID: 10823840 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 167: Arthritis Rheum. 2000 May;43(5):1145-55. Extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 mitogen-activated protein kinase signaling and activation of activator protein 1 and nuclear factor kappaB transcription factors play central roles in interleukin-8 expression stimulated by monosodium urate monohydrate and calcium pyrophosphate crystals in monocytic cells. Liu R, O'Connell M, Johnson K, Pritzker K, Mackman N, Terkeltaub R. Department of Veterans Affairs Medical Center, University of California, San Diego 92161, USA. OBJECTIVE: Monosodium urate monohydrate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals cause acute gout and pseudogout, respectively. Because acute gout and pseudogout appear to be dependent on interleukin-8 (IL-8)-induced neutrophil ingress, this study was undertaken to define and compare how MSU and CPPD crystals stimulate IL-8 messenger RNA (mRNA) expression in mononuclear phagocytes. METHODS: MSU and CPPD crystal-induced mitogen-activated protein kinase (MAPK) signal transduction and IL-8 transcriptional activation were studied in human monocytic cells, using the THP-1 cell line. RESULTS: MSU and CPPD crystals (0.5 mg/ml) induced activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase 1 (ERK-1)/ERK-2, and p38 MAPK pathways in THP-1 cells. Activation of the ERK-1/ERK-2 pathway was essential for MSU and CPPD crystal-induced IL-8 mRNA expression, whereas the p38 pathway played a greater role in IL-8 mRNA expression in response to CPPD crystals in comparison with MSU crystals. Both crystals induced the binding of nuclear factor kappaB (NF-kappaB), including the NF-kappaB complex c-Rel/RelA, and activator protein 1 (AP-1, including N-terminal phosphorylated c-Jun) to the IL-8 promoter. Both crystals induced transcriptional activation of the IL-8 promoter, which was dependent on activation of c-Rel/RelA and AP-1. Activation of the NF-IL-6 transcription factor played a lesser role. Finally, crystal-induced IL-8 promoter activation was mediated by activation of the ERK-1/ERK-2 pathway, as demonstrated by transfection of dominant-negative raf-1. CONCLUSION: These results indicate that ERK-1/ ERK-2 signaling and transcriptional activation through AP-1 and NF-kappaB are essential for the induction of IL-8 expression in mononuclear phagocytes in response to CPPD and MSU crystals. PMID: 10817569 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 168: J Biol Chem. 2000 Apr 28;275(17):13056-60. PDZ domain-dependent suppression of NF-kappaB/p65-induced Abeta42 production by a neuron-specific X11-like protein. Tomita S, Fujita T, Kirino Y, Suzuki T. Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. It is widely believed that one of the causes of Alzheimer's disease (AD) is the generation and secretion of beta-amyloid (Abeta) from amyloid precursor protein in the brain. Here we report that a transcription factor, NF-kappaB/p65, induces increased secretion of amyloidogenic Abeta42 but not Abeta40. The kappaB motif-dependent production of Abeta42 was suppressed by binding of NF-kappaB/p65 to the PDZ domain of the X11-like protein (X11L), which a human homologue protein of LIN-10. The results suggest that the PDZ domain of X11L can control the ability of NF-kappaB/p65 to induce expression of protein(s) involved in Abeta42 production. The amino acids 161-163 in Rel homology domain (RHD) of NF-kappaB/p65 is important in interaction of NF-kappaB/p65 with X11L. Another subunit NF-kappaB/p50 and heterodimers of p65 and p50 do not bind to X11L. Our finding indicates NF-kappaB and X11L may, in novel way, regulate Abeta production in neuronal cells. Targeting X11L by specific therapy may provide the possibility to control the progression of AD. PMID: 10777610 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 169: Virus Res. 2000 Mar;67(1):17-30. Epstein-barr virus latent membrane protein (LMP1) induces specific NFkappaB complexes in human T-lymphoid cells. Chien ML, Hammarskjold ML. Department of Microbiology, State University of New York at Buffalo, Buffalo, NY 14214, USA. The Epstein-Barr virus (EBV) latent membrane protein (LMP1) is believed to play a crucial role in oncogenesis mediated by this virus. We and others previously showed that LMP1 can induce NFkappaB activity in several non-lymphoid cells and B-lymphoid cell lines. Here we show that LMP1 is also able to efficiently induce NFkappaB in human T-lymphoid and monocytic cells. Specific NFkappaB complexes were detected in the nuclei of transfected Jurkat cells using gel mobility shift assays and Western blot analyses. Using antibodies, we demonstrated that these complexes contain NFkappaB subunits NFkB1, NFkB2, RelA and c-Rel. Our results also showed that the NFkappaB complexes induced by LMP1 are able to bind to the NFkappaB consensus sequence in the promoter of the interleukin-2alpha receptor gene and induce expression from a minimal promoter linked to four tandem copies of this sequence. This suggests a possible mechanism by which LMP1 could induce T-cell activation and proliferation. PMID: 10773315 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 170: Biochim Biophys Acta. 2000 Apr 25;1491(1-3):1-6. Growth-phase regulation of the Escherichia coli thioredoxin gene. Lim CJ, Daws T, Gerami-Nejad M, Fuchs JA. Division of Life Sciences, Kangwon National University, Chuncheon, South Korea. cjlim@cc.kangwon.ac.kr The two promoters of Escherichia coli trxA gene were separately cloned into pKO100 as well as pJEL170. Galactokinase expression in cells containing the pKO100 derivatives was found to be negatively correlated with growth rate and was 6- to 20-fold higher in stationary cultures than in exponential cultures. The expression of trxA-galK was induced by amino acid starvation in a RelA(+) strain but not in an isogenic Rel(-) strain indicating that the control involves guanosine 3',5'-bispyrophosphate (ppGpp). RpoS, which appears to be essential for expression of most stationary phase expressed genes, is not required for trxA expression. Increased expression of relA, which increases ppGpp concentration, increases trxA expression. PMID: 10760563 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 171: J Biol Chem. 2000 May 26;275(21):15601-4. L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis. Anto RJ, Maliekal TT, Karunagaran D. Division of Cancer Biology, Rajiv Gandhi Center for Biotechnology, Thiruvananthapuram, Kerala-695014, India. Curcumin (diferuloyl methane), the yellow pigment in turmeric (Curcuma longa), is a potent chemopreventive agent. Curcumin induces apoptosis of several, but not all, cancer cells. Many cancer cells protect themselves against apoptosis by activating nuclear factor-kappaB (NF-kappaB)/Rel, a transcription factor that helps in cell survival. Signal-induced activation of NF-kappaB is known to be inhibited by curcumin. To understand the role of NF-kappaB in curcumin-induced apoptosis, we stably transfected relA gene encoding the p65/RelA subunit of NF-kappaB, into l-929 cells (mouse fibrosarcoma) and the relA-transfected cells were resistant to varying doses of curcumin (10(-6)-10(-4) m), whereas the parental cells underwent apoptosis in a time- and dose-dependent manner. The relA-transfected cells showed constitutive NF-kappaB DNA binding activity that could not be inhibited by curcumin and did not show nuclear condensation and DNA fragmentation upon treatment with curcumin. When a super-repressor form of IkappaB-alpha (known to inhibit NF-kappaB) was transfected transiently into relA-transfected cells, the cells were no longer resistant to curcumin. Our results highlight a critical anti-apoptotic role for NF-kappaB in curcumin-induced apoptosis. PMID: 10747850 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 172: Int Immunol. 2000 Apr;12(4):547-54. Stimulation of Fc gamma R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving I kappa B-alpha degradation and formation of p50/p65 NF-kappa B/Rel complexes. Alonso A, Bayon Y, Renedo M, Crespo MS. Instituto de Biologia y Genetica Molecular, Consejo Superior de Investigaciones Cientificas, Facultad de Medicina, 47005 Valladolid, Spain. THP-1 monocytic/macrophage cells were stimulated via their FcgammaR receptors with insoluble aggregates of human IgG and the production of the C-C chemokine monocyte chemoattractant protein (MCP)-1 assayed. A dose- and time-dependent production of MCP-1 comparable to that produced by the most potent agonists could be detected in the culture medium by a sensitive ELISA assay. This was accompanied by a parallel activation of the transcription factor NF-kappaB as judged from both the appearance of kappaB-binding activity containing p50/p65 NF-kappaB/Rel complexes in the nuclear extract and the disappearance of the NF-kappaB inhibitor IkappaB-alpha in the cell lysate. In contrast, IkappaB-beta and IkappaB-epsilon expression was not modified, thus pointing to the occurrence of a selective degradation of IkappaB-alpha under those conditions. Attempts to modulate MCP-1 production with compounds that display inhibitory effects on the activation of NF-kappaB such as the proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal, the antioxidant pyrrolidine dithiocarbamate and the salicylate derivative 2-hydroxy-4-trifluoromethylbenzoic acid showed a parallel effect on both MCP-1 production and NF-kappaB activation, thus pointing to the involvement of kappaB-binding sites on the transcriptional regulation of MCP-1 production. Our findings suggest the existence in monocytic cells of a signaling mechanism initiated by cross-linking of low-affinity FcgammaR, most likely of the FcgammaRII family since THP-1 cells do not express FcgammaRIII receptors, that involves activation of NF-kappaB associated to the proteolytic degradation of IkappaB-alpha and leads to the transcriptional up-regulation of MCP-1. PMID: 10744656 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 173: Mol Cell Biol. 2000 Apr;20(8):2687-95. The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L). Chen C, Edelstein LC, Gelinas C. Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5638, USA. The transcription factors of the Rel/NF-kappaB family are key regulators of immune and inflammatory responses and contribute to lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities of the Rel/NF-kappaB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-kappaB control. Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-kappaB raised the issue of whether NF-kappaB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate that NF-kappaB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates the expression of Bcl-x(L). While Bcl-x(L) was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-kappaB factors also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-kappaB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-kappaB activity. Functional analysis of the bcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-kappaB directly regulates the expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-x(L) and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with aberrant Rel/NF-kappaB activity. PMID: 10733571 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 174: J Cell Biochem. 2000 Mar;77(2):221-33. Activation of a rel-A/CEBP-beta-related transcription factor heteromer by PGG-glucan in a murine monocytic cell line. Adams DS, Nathans R, Pero SC, Sen A, Wakshull E. Department of Biology/Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA. dadams@wpi.edu PGG-Glucan is a soluble beta-glucan immunomodulator that enhances a variety of leukocyte microbicidal activities without activating inflammatory cytokines. Although several different cell surface receptors for soluble (and particulate) beta-glucans have been described, the signal transduction pathway(s) used by these soluble ligands have not been elucidated. Previously we reported that PGG-Glucan treatment of mouse BMC2.3 macrophage cells activates a nuclear factor kappa-B-like (NF-kappaB) transcription factor complex containing subunit p65 (rel-A) attached to an unidentified cohort. In this study, we identify the cohort to be a non-rel family member: a CCAAT enhancer-binding protein-beta (C/EBP-beta)-related molecule with an apparent size of 48 kDa, which is a different protein than the previously identified C/EBP-beta p34 also present in these cells. C/EBP-beta is a member of the bZIP family whose members have previously been shown to interact with rel family members. This rel/bZIP heteromer complex activated by PGG-Glucan is different from the p65/p50 rel/rel complex induced in these cells by lipopolysaccharide (LPS). Thus, our data demonstrate that PGG-Glucan uses signal transduction pathways different from those used by LPS, which activates leukocyte microbicidal activities and inflammatory cytokines. We further show that heteromer activation appears to use protein kinase C (PKC) and protein tyrosine kinase (PTK) pathways, but not mitogen-activated protein kinase p38. Inhibitor kappa-B-alpha (IkappaB-alpha) is associated with the heteromer; this association decreases after PGG-Glucan treatment. These data are consistent with a model whereby treatment of BMC2.3 cells with PGG-Glucan activates IkappaB-alpha via PKC and/or PTK pathways, permitting translocation of the rel-A/CEBP-beta heteromer complex to the nucleus and increases its DNA-binding affinity. Copyright 2000 Wiley-Liss, Inc. PMID: 10723089 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 175: J Biol Chem. 2000 Mar 17;275(11):7619-25. The structure of the nuclear factor-kappaB protein-DNA complex varies with DNA-binding site sequence. Menetski JP. Department of Molecular Biology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert, Ann Arbor, Michigan 48105, USA. joseph.menetski@wl.com Transcriptional regulation of many immune responsive genes is under the control of the transcription factor NF-kappaB. This factor is found in cells as a dimer which can contain any two members of the Rel family of proteins (p50, p65, p52, c-Rel, and RelB). The different dimers show distinct preferences for DNA-binding site sequences. To understand the relationship between the DNA binding properties of the dimer forms and transcriptional activation, the physical properties of the complexes of p50 and p65 with DNA have been analyzed. Comparison of apparent DNA binding affinity showed differences in selectivity of DNA-binding site sequence. The ionic strength dependence of apparent binding affinity has shown that the number of ionic interactions in the protein-DNA complex depends on the DNA-binding site sequence and the dimer form, which are consistent with changes in the structure of the protein-DNA complex. Using a fluorescent technique to measure DNA structure changes, protein binding does not appear to alter the structure of the DNA-binding site within the limits of detection. These results are consistent with a change in protein structure that may result in activation differences due to alternative interactions with other transcription proteins. PMID: 10713070 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 176: Oncogene. 2000 Feb 24;19(9):1123-31. Selective activation of NF-kappa B subunits in human breast cancer: potential roles for NF-kappa B2/p52 and for Bcl-3. Cogswell PC, Guttridge DC, Funkhouser WK, Baldwin AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, NC 27599-7295, USA. Members of the NF-kappa B/Rel transcription factor family have been shown recently to be required for cellular transformation by oncogenic Ras and by other oncoproteins and to suppress transformation-associated apoptosis. Furthermore, NF-kappa B has been shown to be activated by several oncoproteins including HER2/Neu, a receptor tyrosine kinase often expressed in human breast cancer. Human breast cancer cell lines, human breast tumors and normal adjacent tissue were analysed by gel mobility shift assay, immunoblotting of nuclear extracts and immunohistochemistry for activation of NF-kappa B. Furthermore, RNA levels for NF-kappa B-activated genes were analysed in order to determine if NF-kappa B is functionally active in human breast cancer. Our data indicate that the p65/RelA subunit of NF-kappa B is activated (i.e., nuclear) in breast cancer cell lines. However, breast tumors exhibit an absence or low level of nuclear p65/RelA but show activated c-Rel, p50 and p52 as compared to nontumorigenic adjacent tissue. Additionally, the I kappa B homolog Bcl-3, which functions to stimulate transcription with p50 or p52, was also activated in breast tumors. There was no apparent correlation between estrogen receptor status and levels of nuclear NF-kappa B complexes. Transcripts of NF-kappa B-regulated genes were found elevated in breast tumors, as compared to adjacent normal tissue, indicating functional NF-kappa B activity. These data suggest a potential role for a subset of NF-kappa B and I kappa B family proteins, particularly NF-kappa B/p52 and Bcl-3, in human breast cancer. Additionally, the activation of functional NF-kappa B in these tumors likely involves a signal transduction pathway distinct from that utilized by cytokines. PMID: 10713699 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 177: Am J Pathol. 2000 Mar;156(3):997-1007. Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double knockout mice. Rosenfeld ME, Prichard L, Shiojiri N, Fausto N. Department of Pathology, University of Washington, Seattle, Washington 98195, USA. Mice deficient in the nuclear factor kappaB (NF-kappaB)-transactivating gene RelA (p65) die at embryonic days 14-15 with massive liver apoptosis. In the adult liver, activation of the NF-kappaB heterodimer RelA/p50 can cause hepatocyte proliferation, apoptosis, or the induction of acute-phase response genes. We examined, during wild-type fetal liver development, the expression of the Rel family member proteins, as well as other proteins known to be important for NF-kappaB activation. We found these proteins and active NF-kappaB complexes in the developing liver from at least 2 days before the onset of lethality observed in RelA knockouts. This suggests that the timing of NF-kappaB activation is not related to the timing of lethality. We therefore hypothesized that, in the absence of RelA, embryos were sensitized to tumor necrosis factor (TNF) receptor 1 (TNFR-1)-mediated apoptosis. Thus, we generated mice that were deficient in both RelA and TNFR-1 to determine whether apoptotic signaling through TNFR-1 was responsible for the lethal phenotype. RelA/TNFR-1 double knockout mice survived embryonic development and were born with normal livers without evidence of increased hepatocyte apoptosis. These animals became runted shortly after birth and survived an average of 10 days, dying from acute hepatitis with an extensive hepatic infiltration of immature neutrophils. We conclude that neither RelA nor TNFR-1 is required for liver development and that RelA protects the embryonic liver from TNFR-1-mediated apoptotic signals. However, the absence of both TNFR-1 signaling and RelA activity in newborn mice makes these animals susceptible to endogenous hepatic infection. PMID: 10702415 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 178: Mol Cell Biol. 2000 Mar;20(6):2269-84. Cytoplasmic sequestration of rel proteins by IkappaBalpha requires CRM1-dependent nuclear export. Tam WF, Lee LH, Davis L, Sen R. Rosenstiel Basic Medical Sciences Research Center, Waltham, Massachusetts 02454, USA. Rel and IkappaB protein families form a complex cellular regulatory network. A major regulatory function of IkappaB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IkappaB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-kappaB-dependent gene transcription, as well as termination of inducible transcription. We show that IkappaBalpha shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IkappaBalpha but not in that of IkappaBbeta or IkappaBepsilon. By reconstituting major aspects of NF-kappaB-IkappaB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IkappaBalpha requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IkappaBalpha in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IkappaBalpha is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IkappaBalpha association, from where these complexes must be exported in order to create the cytoplasmic pool. PMID: 10688673 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 179: J Virol. 2000 Mar;74(6):2655-62. Human T-cell leukemia virus type 2 tax mutants that selectively abrogate NFkappaB or CREB/ATF activation fail to transform primary human T cells. Ross TM, Narayan M, Fang ZY, Minella AC, Green PL. Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363, USA. Human T-cell leukemia virus (HTLV) Tax protein has been implicated in the HTLV oncogenic process, primarily due to its pleiotropic effects on cellular genes involved in growth regulation and cell cycle control. To date, several approaches attempting to correlate Tax activation of the CREB/activating transcription factor (ATF) or NFkappaB/Rel transcriptional activation pathway to cellular transformation have yielded conflicting results. In this study, we use a unique HTLV-2 provirus (HTLV(c-enh)) that replicates by a Tax-independent mechanism to directly assess the role of Tax transactivation in HTLV-mediated T-lymphocyte transformation. A panel of well-characterized tax-2 mutations is utilized to correlate the respective roles of the CREB/ATF or NFkappaB/Rel signaling pathway. Our results demonstrate that viruses expressing tax-2 mutations that selectively abrogate NFkappaB/Rel or CREB/ATF activation display distinct phenotypes but ultimately fail to transform primary human T lymphocytes. One conclusion consistent with our results is that the activation of NFkappaB/Rel provides a critical proliferative signal early in the cellular transformation process, whereas CREB/ATF activation is required to promote the fully transformed state. However, complete understanding will require correlation of Tax domains important in cellular transformation to those Tax domains important in the modulation of gene transcription, cell cycle control, induction of DNA damage, and other undefined activities. PMID: 10684280 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 180: J Biol Chem. 2000 Feb 18;275(7):4719-25. Inhibition of the RelA(p65) NF-kappaB subunit by Egr-1. Chapman NR, Perkins ND. Department of Biochemistry, Division of Gene Expression and Regulation, MSI/WTB Complex, Dow Street, University of Dundee, Dundee, DD1 5EH Scotland, United Kingdom. Induction of transcription from the human immunodeficiency virus 1 long terminal repeat by the RelA (p65) NF-kappaB subunit has been shown to be dependent upon an interaction with the zinc finger DNA-binding domain of Sp1. It was unknown, however, whether NF-kappaB could also interact with other zinc finger-containing transcription factors. In this study we demonstrate that the early growth response transcription factor Egr-1, whose DNA-binding domain shares a high degree of homology with that of Sp1, can also interact with RelA in vitro and regulate NF-kappaB transcriptional activity in vivo. Similar to the interaction with Sp1, the Rel homology domain of RelA interacts with the zinc finger domain of Egr-1. Surprisingly, and in contrast to Sp1, Egr-1 specifically represses RelA transcriptional activity through its zinc finger domain. Moreover, the interaction between RelA and the Egr-1 zinc fingers is mutually exclusive with DNA binding suggesting a model in which Egr-1 directly sequesters NF-kappaB from its target promoters. Because Egr-1 is induced by many of the same stimuli that activate NF-kappaB, this novel transcriptional regulatory mechanism has many implications for the involvement of both factors in cellular processes such as apoptosis and the response to stress and infection. PMID: 10671503 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 181: Shock. 2000 Feb;13(2):85-91. NF-kappaB regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome. Moine P, McIntyre R, Schwartz MD, Kaneko D, Shenkar R, Le Tulzo Y, Moore EE, Abraham E. Departement d'Anesthesie Reanimation Chirurgicale, CHU de Bicetre, Universite Paris Sud, France. Activation of the nuclear regulatory factor NF-kappaB occurs in the lungs of patients with the acute respiratory distress syndrome (ARDS) and may contribute to the increased expression of immunoregulatory cytokines and other proinflammatory mediators in this setting. Because of the important role that NF-kappaB activation appears to play in the development of acute lung injury, we examined cytoplasmic and nuclear NF-kapppaB counterregulatory mechanisms, involving IkappaB proteins, in alveolar macrophages obtained from 7 control patients without lung injury and 11 patients with established ARDS. Cytoplasmic levels of the NF-kappaB subunits p50, p65, and c-Rel were significantly decreased in alveolar macrophages from patients with ARDS, consistent with enhanced migration of liberated NF-kappaB dimers from the cytoplasm to the nucleus. Cytoplasmic and nuclear levels of IkappaBalpha were not significantly altered in alveolar macrophages from patients with established ARDS, compared with controls. In contrast, nuclear levels of Bcl-3 were significantly decreased in patients with ARDS compared with controls (P = 0.02). No IkappaBgamma, IkappaBbeta, or p105 proteins were detected in the cytoplasm of alveolar macrophages from control patients or patients with ARDS. The presence of activated NF-kappaB in alveolar macrophages from patients with established ARDS implies the presence of an ongoing stimulus for NF-kappaB activation. In this setting, appropriate counterregulatory mechanisms to normalize nuclear levels of NF-kappaB and to suppress NF-kappaB-mediated transcription, such as increased cytoplasmic and nuclear IkappaBalpha levels or decreased Bcl-3 levels, appeared to be induced. Nevertheless, even though counterregulatory mechanisms to NF-kappaB activation are activated in lung macrophages of patients with ARDS, NF-kappaB remains activated. These results suggest that fundamental abnormalities in transcriptional mechanisms involving NF-kappaB and important in the inflammatory response occur in the lungs of patients with ARDS. Publication Types: Clinical Trial Controlled Clinical Trial PMID: 10670837 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 182: Biochem Biophys Res Commun. 2000 Feb 5;268(1):99-105. Differential expression of the transcription factor NF-kappaB during human mononuclear phagocyte differentiation to macrophages and dendritic cells. Ammon C, Mondal K, Andreesen R, Krause SW. Department of Hematology, University of Regensburg, Regensburg, D-93042, Germany. An important role for the Rel/NF-kappaB family of transcription factors in the differentiation process of dendritic cells (DC) and macrophages (MAC) was recently suggested by a number of mouse knockout studies but only little information is available for defined populations of human cells. To investigate the role of individual NF-kappaB proteins [p50, p52, p65 (RelA), RelB] in the differentiation of monocyte-derived cell types we analyzed and compared the expression pattern and DNA binding activity of NF-kappaB members in human monocytes (MO), MO-derived MAC, and MO-derived DC. Constitutive expression of p65 and RelB mRNA was found in MO and no significant regulation was observed during differentiation of MO into MAC or immature DC. Only during lipopolysaccharide-induced terminal differentiation of DC was a marked increase in RelB mRNA detected. In DNA binding assays performed with nuclear extracts from blood MO, p50/p50 homodimers were mainly detected, whereas complexes containing p50/RelB and p50/p65 heterodimers were less abundant. DNA-bound protein complexes containing p50/RelB and p50/p65 increased and additional p65/p65 complexes appeared during differentiation of MO into either MAC or immature DC. A strong increase in complexes containing p50/RelB was observed during terminal differentiation of DC. Therefore, gradual differences in the DNA binding activities of different NF-kappaB homo- and heterodimers correlate with differentiation stages of MO, MAC, and DC and are probably important for the biological role of these cells. Copyright 2000 Academic Press. PMID: 10652220 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 183: Oncogene. 1999 Nov 22;18(49):6938-47. Aberrant rel/nfkb genes and activity in human cancer. Rayet B, Gelinas C. Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, NJ 08854-5638, USA. Rel/NF-kappaB transcription factors are key regulators of immune, inflammatory and acute phase responses and are also implicated in the control of cell proliferation and apoptosis. Remarkable progress has been made in understanding the signal transduction pathways that lead to the activation of Rel/NF-kappaB factors and the consequent induction of gene expression. Evidence linking deregulated Rel/NF-kappaB activity to oncogenesis in mammalian systems has emerged in recent years, consistent with the acute oncogenicity of the viral oncoprotein v-Rel in animal models. Chromosomal amplification, overexpression and rearrangement of genes coding for Rel/NF-kappaB factors have been noted in many human hematopoietic and solid tumors. Persistent nuclear NF-kappaB activity was also described in several human cancer cell types, as a result of constitutive activation of upstream signaling kinases or mutations inactivating inhibitory IkappaB subunits. Studies point to a correlation between the activation of cellular gene expression by Rel/NF-kappaB factors and their participation in the malignant process. Experiments implicating NF-kappaB in the control of the apoptotic response also support a role in oncogenesis and in the resistance of tumor cells to chemotherapy. This review focuses on the status of the rel, nfkb and ikb genes and their activity in human tumors and their association with the onset or progression of malignancies. Publication Types: Review Review, Tutorial PMID: 10602468 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 184: Oncogene. 1999 Nov 22;18(49):6910-24. Control of apoptosis by Rel/NF-kappaB transcription factors. Barkett M, Gilmore TD. Boston University, Biology Department, 5 Cummington Street, Boston, Massachusetts, MA 02215, USA. Apoptosis is a physiological process critical for organ development, tissue homeostasis, and elimination of defective or potentially dangerous cells in complex organisms. Apoptosis can be initiated by a wide variety of stimuli, which activate a cell suicide program that is constitutively present in most vertebrate cells. In diverse cell types, Rel/NF-kappaB transcription factors have been shown to have a role in regulating the apoptotic program, either as essential for the induction of apoptosis or, perhaps more commonly, as blockers of apoptosis. Whether Rel/NF-kappaB promotes or inhibits apoptosis appears to depend on the specific cell type and the type of inducer. An understanding of the role of Rel/NF-kappaB transcription factors in controlling apoptosis may lead to the development of therapeutics for a wide variety of human diseases, including neurodegenerative and immune diseases, and cancer. Publication Types: Review Review, Tutorial PMID: 10602466 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 185: Oncogene. 1999 Nov 22;18(49):6896-909. Diverse agents act at multiple levels to inhibit the Rel/NF-kappaB signal transduction pathway. Epinat JC, Gilmore TD. Boston University, Biology Department, 5 Cummington Street, Boston, Massachusetts, MA 02215, USA. Rel/NF-kappaB transcription factors regulate several important physiological processes, including developmental processes, inflammation and immune responses, cell growth, cancer, apoptosis, and the expression of certain viral genes. Therefore, they have also been sought-after molecular targets for pharmacological intervention. As details of the Rel/NF-kappaB signal transduction pathway are revealed, it is clear that modulators of this pathway can act at several levels. Inhibitors of the Rel/NF-kappaB pathway include a variety of natural and designed molecules, including anti-oxidants, proteasome inhibitors, peptides, small molecules, and dominant-negative or constitutively active polypeptides in the pathway. Several of these molecules act as general inhibitors of Rel/NF-kappaB induction, whereas others inhibit specific pathways of induction. Inhibitors of Rel/NF-kappaB are likely to gain stature as treatments for certain cancers and neurodegenerative and inflammatory diseases. Publication Types: Review Review, Tutorial PMID: 10602465 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 186: Oncogene. 1999 Nov 22;18(49):6888-95. Genetic approaches in mice to understand Rel/NF-kappaB and IkappaB function: transgenics and knockouts. Gerondakis S, Grossmann M, Nakamura Y, Pohl T, Grumont R. The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Rel/NF-kappaB transcription factors have been implicated in regulating a wide variety of genes important in cellular processes that include cell division, cell survival, differentiation and immunity. Here genetic models in which various Rel/NF-kappaB and IkappaB proteins have either been over-expressed or deleted in mice will be reviewed. Although expressed fairly ubiquitously, homozygous disruption of individual Rel/NF-kappaB genes generally affects the development of proper immune cell function. One exception is rela, which is essential for embryonic liver development. The disruption of genes encoding the individual subunits of the IkappaB kinase, namely IKKalpha and IKKbeta, has demonstrated that IKKbeta transmits the response to most common NF-kappaB inducing agents, whereas IKKalpha has an unexpected role in keratinocyte differentiation. Future studies will no doubt focus on the effect of multiple gene disruptions of members of this signaling pathway, on tissue-specific disruptions of these genes, and on the use of these mice as models for human diseases. Publication Types: Review Review, Tutorial PMID: 10602464 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 187: Oncogene. 1999 Nov 22;18(49):6875-87. Control of development and immunity by rel transcription factors in Drosophila. Govind S. Department of Biology, City College and The Graduate Center of The City University of New York, 138th Street and Convent Avenue, New York, NY 10031, USA. The Drosophila Rel/NF-kappaB transcription factors - Dorsal, Dif, and Relish - control several biological processes, including embryonic pattern formation, muscle development, immunity, and hematopoiesis. Molecular-genetic analysis of 12 mutations that cause embryonic dorsal/ventral patterning defects has defined the steps that control the formation of this axis. Regulated activation of the Toll receptor leads to the establishment of a gradient of nuclear Dorsal protein, which in turn governs the subdivision of the axis and specification of ventral, lateral and dorsal fates. Phenotypic analysis of dorsal-ventral embryonic mutants and the characterization of the two other fly Rel proteins, Dif and Relish, have shown that the intracellular portion of the Toll to Cactus pathway also controls the innate immune response in Drosophila. Innate immunity and hematopoiesis are regulated by analogous Rel/NF-kappaB-family pathways in mammals. The elucidation of the complex regulation and diverse functions of Drosophila Rel proteins underscores the relevance of basic studies in Drosophila. Publication Types: Review Review, Tutorial PMID: 10602463 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 188: Oncogene. 1999 Nov 22;18(49):6845-52. Regulation of DNA binding by Rel/NF-kappaB transcription factors: structural views. Chen FE, Ghosh G. Department of Biology, University of California - San Diego, 9500 Gilman Drive, MC 0359, La Jolla, California, CA 92093-0359, USA. Rel/NF-kappaB transcription factors form homo- and heterodimers with different DNA binding site specificities and DNA binding affinities. Several intracellular pathways evoked by a wide range of biological factors and environmental conditions can lead to the activation of Rel/NF-kappaB dimers by signaling degradation of the inhibitory IkappaB protein. In the nucleus Rel/NF-kappaB dimers modulate the expression of a variety of genes including those encoding cytokines, growth factors, acute phase response proteins, immunoreceptors, other transcription factors, cell adhesion molecules, viral proteins and regulators of apoptosis. The primary focus of this review is on structural and functional aspects of Rel/NF-kappaB:DNA complexes and their formation. The salient features of the Rel/NF-kappaB dimer:DNA structure are described, as are modes of transcriptional regulation by phosphorylation, altered DNA binding properties, varying protein conformations, and interactions with IkappaB proteins. Publication Types: Review Review, Tutorial PMID: 10602460 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 189: Oncogene. 1999 Nov 22;18(49):6842-4. The Rel/NF-kappaB signal transduction pathway: introduction. Gilmore TD. Biology Department, Boston University, 5 Cummington Street, Boston, Massachusetts, MA 02215-2406, USA. Publication Types: Review Review, Tutorial PMID: 10602459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 190: Int J Biochem Cell Biol. 1999 Oct;31(10):1209-19. New insights into the roles of ReL/NF-kappa B transcription factors in immune function, hemopoiesis and human disease. Grossmann M, Nakamura Y, Grumont R, Gerondakis S. Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia. In mammals, Rel/NF-kappa B proteins are a small family of transcription factors which serve as pivotal regulators of immune, inflammatory and acute phase responses. Pathways leading to the activation of Rel/NF-kappa B have recently been dissected in some detail and shown to converge on a unique high molecular weight cytoplasmic complex that includes several kinases and regulatory molecules. Moreover, gene targeting experiments have identified novel roles for Rel/NF-kappa B proteins in the development and maturation of hemopoietic precursors as well as in the function of mature cells in the immune system. These include regulating the cell cycle, controlling cell survival and providing a link between the innate and adaptive immune systems. Since the dysregulation of Rel/NF-kappa B function is associated with various pathologies including inflammatory and neoplastic disease, new insights into the role of Rel/NF-kappa B in human disease may provide a basis for therapeutic strategies in the treatment of chronic inflammatory diseases and certain malignancies. Publication Types: Review Review, Tutorial PMID: 10582348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 191: EMBO J. 1999 Dec 1;18(23):6682-93. An N-terminal nuclear export signal is required for the nucleocytoplasmic shuttling of IkappaBalpha. Johnson C, Van Antwerp D, Hope TJ. Infectious Disease Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA. The potent transcriptional activities of Rel/NF-kappaB proteins are regulated in the cytoplasm and nucleus by the inhibitor, IkappaBalpha. The mechanism, by which IkappaBalpha can either sequester NF-kappaB in the cytoplasm or act as a nuclear post-induction repressor of NF-kappaB, is uncertain. We find that IkappaBalpha shuttles continuously between the nucleus and cytoplasm. This shuttling requires a previously unidentified CRM1-dependent nuclear export signal (NES) located within the N-terminal domain of IkappaBalpha at amino acids 45-55. Deletion or mutation of the N-terminal NES results in nuclear localization of IkappaBalpha. NF-kappaB (p65) association with IkappaBalpha affects steady-state localization but does not inhibit its shuttling. Endogenous complexes of IkappaBalpha-NF-kappaB shuttle and will accumulate in the nucleus when CRM1 export is blocked. We find TNFalpha can activate the nuclear IkappaBalpha-NF-kappaB complexes by the classical mechanism of proteasome-mediated degradation of IkappaBalpha. These studies reveal a more dynamic nucleocytoplasmic distribution for IkappaBalpha and NF-kappaB suggesting previously unknown strategies for regulating this ubiquitous family of transcription activators. PMID: 10581242 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 192: J Immunol. 1999 Nov 15;163(10):5656-65. Substance P activates coincident NF-AT- and NF-kappa B-dependent adhesion molecule gene expression in microvascular endothelial cells through intracellular calcium mobilization. Quinlan KL, Naik SM, Cannon G, Armstrong CA, Bunnett NW, Ansel JC, Caughman SW. Department of Dermatology, Emory Skin Diseases Research Core Center, Emory University School of Medicine, Atlanta, GA 30322, USA. Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the -191/-170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-alpha), and NF-kappa B (p65/p50) to tandem NF-kappa B binding sites at -76/-52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-kappa B. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-kappa B activation and binding. These data demonstrate coincident activation of NF-AT and NF-kappa B via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses. PMID: 10553096 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 193: Virology. 1999 Oct 10;263(1):128-38. Lack of responsiveness of a nuclear factor-kappaB-regulated promoter to transactivation by human immunodeficiency virus 1 Tat in HeLa cells. Kelly GD, Morris CB, Offermann MK. Department of Medicine, Emory University, Atlanta, Georgia 30322, USA. Transcriptional activation by Tat protein is in large part dependent on interactions with the TAR RNA element located in the 5'-untranslated region of all human immunodeficiency virus type 1 (HIV-1) transcripts. In addition, Tat has been shown to induce nuclear translocation of nuclear factor-kappaB (NF-kappaB), potentially contributing to gene induction. The NF-kappaB responsive reporter construct, (PRDII)(4)-CAT, was used to explore transcription resulting from NF-kappaB activated by Tat. Tat did not activate (PRDII)(4)-CAT, whereas (PRDII)(4)-CAT was highly responsive to either transfected Rel A or to tumor necrosis factor-alpha (TNF-alpha). Despite its inability to directly induce, Tat enhanced the responsiveness of (PRDII)(4)-CAT to either transfected Rel A or to TNF-alpha by approximately 2.5-fold. High levels of CAT activity were seen with HIV-LTR-derived reporters that contained kappaB and TAR elements in response to transfected Tat in the absence of either transfected Rel A or exogenous TNF-alpha, and overexpression of IkappaBalpha with Tat inhibited CAT activity by 60% to 80%, suggesting that some activation of NF-kappaB by Tat was occurring. HIV-LTR reporter activities were enhanced three fold to sixfold compared with Tat alone when additional NF-kappaB was provided by transfection or by activation with TNF-alpha. These data indicate that Tat is unable to activate some NF-kappaB-responsive promoters but is able to synergize with NF-kappaB in the activation of both HIV-derived and non-HIV-derived promoters. Copyright 1999 Academic Press. PMID: 10544088 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 194: Gene Expr. 1999;8(1):1-18. A multiprotein complex consisting of the cellular coactivator p300, AP-1/ATF, as well as NF-kappaB is responsible for the activation of the mouse major histocompatibility class I (H-2K(b)) enhancer A. Brockmann D, Putzer BM, Lipinski KS, Schmucker U, Esche H. Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Germany. dieter.brockmann@uni-essen.de Major histocompatibility complex (MHC) class I genes encode highly polymorphic antigens that play an essential role in a number of immunological processes. Their expression is activated in response to a variety of signals and is mediated through several promoter elements among which the enhancer A is one of the key control regions. It contains binding sites for several transcription factors, for example: (i) a well-characterized binding site for rel/NF-kappaB transcription factors in its 3'-end (the H2TF1 or kappaB1 element), (ii) a second kappaB site (the kappaB2 element), which is located immediately adjacent 5' to the H2TF1 element and which is recognized by p65/relA in the human HLA system, and (iii) an AP-1/ATF recognition sequence in the 5' end (EnA-TRE). Here we demonstrate that latter element is bound by at least two distinct heterodimers of the AP-1/ATF transcription factor family, namely c-Jun/ATF-2 and c-Jun/Fra2. Moreover, our data reveal that the enhancer A is simultaneously bound by AP-1/ATF and rel/NF-kappaB transcription factors and that the cellular coactivator p300, which enhances enhancer A-driven reporter gene expression if cotransfected, is recruited to the enhancer A through this multiprotein complex. In contrast to the complete enhancer A, neither the EnA-TRE nor the H2TF1 element on their own are able to confer activation on a heterologous promoter in response to the phorbol ester tumor promoter TPA or the cytokine TNFalpha. Moreover, deletion of any one of the enhancer A control elements results in a dramatic loss of its inducibility by TNFalpha, and point mutations in either the EnA-TRE or the H2TF1 element lead to the loss of AP-1/ATF or NF-kappaB binding, respectively, and to the loss of enhancer A inducibility. Therefore, we conclude that the enhancer A is synergistically activated through a multiprotein complex containing AP-1/ATF, NF-kappaB transcription factors as well as the cellular coactivator p300. PMID: 10543727 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 195: DNA Cell Biol. 1999 Oct;18(10):751-61. NF-kappaB inhibits expression of the alpha1(I) collagen gene. Rippe RA, Schrum LW, Stefanovic B, Solis-Herruzo JA, Brenner DA. Department of Medicine, The University of North Carolina, Chapel Hill 27955-7038, USA. rarippe@med.unc.edu Fibrosis results from an increase in the synthesis and deposition of type I collagen. Fibrosis is frequently associated with inflammation, which is accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and activation of the transcription factor NF-kappaB. However, several agents known to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and TNFalpha, result in decreased expression of type I collagen. Therefore, we directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells (HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50, p65, and c-Rel expression plasmids with collagen reporter gene plasmids demonstrated a strong inhibitory effect on transcription of the collagen gene promoter. Dose-response curves showed that p65 was a stronger inhibitor of collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition 90%). Transient transfections with reporter gene plasmids containing one or two Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on the activity of these reporter genes, suggesting that the inhibitory effects of NF-kappaB p65 are mediated through the critical Spl binding sites in the alpha1(I) collagen gene promoter. Cotransfection experiments using either a super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene. Together, these results demonstrate that NF-kappaB decreases transcription of the alpha1(I) collagen gene. PMID: 10541434 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 196: J Immunol. 1999 Nov 1;163(9):5116-24. Preferential role for NF-kappa B/Rel signaling in the type 1 but not type 2 T cell-dependent immune response in vivo. Aronica MA, Mora AL, Mitchell DB, Finn PW, Johnson JE, Sheller JR, Boothby MR. Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical School, Nashville, TN 37232, USA. T cell function is a critical determinant of immune responses as well as susceptibility to allergic diseases. Activated T cells can differentiate into effectors whose cytokine profile is limited to type 1 (IFN-gamma-dominant) or type 2 (IL-4-, IL-5-dominant) patterns. To investigate mechanisms that connect extracellular stimuli with the regulation of effector T cell function, we have measured immune responses of transgenic mice whose NF-kappa B/Rel signaling pathway is inhibited in T cells. Surprisingly, these mice developed type 2 T cell-dependent responses (IgE and eosinophil recruitment) in a model of allergic pulmonary inflammation. In contrast, type 1 T cell responses were severely impaired, as evidenced by markedly diminished delayed-type hypersensitivity responses, IFN-gamma production, and Ag-specific IgG2a levels. Taken together, these data indicate that inhibition of NF-kappa B can lead to preferential impairment of type 1 as compared with type 2 T cell-dependent responses. PMID: 10528218 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 197: Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):11848-53. The combined absence of the transcription factors Rel and RelA leads to multiple hemopoietic cell defects. Grossmann M, Metcalf D, Merryfull J, Beg A, Baltimore D, Gerondakis S. The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Individual Rel/NF-kappaB transcription factors, although dispensable for the development and maturation of most hemopoietic cells, are critical regulators of normal immune function. Redundancy among these proteins prompted us to examine the role of Rel and RelA in hemopoiesis by using mice that lack both subunits. Because of the death of double-mutant fetuses at day 13.5 of gestation (E13.5), E12 fetal liver hemopoietic progenitors were used for in vitro cultures and for repopulating stem cell studies in lethally irradiated normal recipient mice. Most striking, Rel/RelA-deficient hemopoietic precursors failed to promote the survival of myeloablated mice. This phenotype was associated with several defects including a reduction of spleen colony-forming unit progenitors, impaired erythropoiesis, and a deregulated expansion of granulocytes. In vitro progenitor assays also revealed that Rel or RelA serves an antiapoptotic role during monocyte differentiation. Despite the combined loss of Rel and RelA leading to these hemopoietic defects, c-rel(-/-)rela(-/-) stem cells contributed to the development of all lineages in mice engrafted with double-mutant fetal liver cells and normal bone marrow cells, albeit in a reduced fashion compared with controls. Collectively, these data indicate the loss of Rel and RelA does not appear to affect pluripotent stem cells; rather, Rel and RelA serve redundant functions in regulating differentiation and survival of committed progenitors in multiple hemopoietic lineages. PMID: 10518539 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 198: Infect Immun. 1999 Oct;67(10):5434-40. Infection of endothelial cells with Trypanosoma cruzi activates NF-kappaB and induces vascular adhesion molecule expression. Huang H, Calderon TM, Berman JW, Braunstein VL, Weiss LM, Wittner M, Tanowitz HB. Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.Huangh@aecom.yu.edu Transcriptional activation of vascular adhesion molecule expression, a major component of an inflammatory response, is regulated, in part, by the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors. We therefore determined whether Trypanosoma cruzi infection of endothelial cells resulted in the activation of NF-kappaB and the induction or increased expression of adhesion molecules. Human umbilical vein endothelial cells (HUVEC) were infected with trypomastigotes of the Tulahuen strain of T. cruzi. Electrophoretic mobility shift assays with an NF-kappaB-specific oligonucleotide and nuclear extracts from T. cruzi-infected HUVEC (6 to 48 h postinfection) detected two major shifted complexes. Pretreatment with 50x cold NF-kappaB consensus sequence abolished both gel-shifted complexes while excess SP-1 consensus sequence had no effect. These data indicate that nuclear extracts from T. cruzi-infected HUVEC specifically bound to the NF-kappaB consensus DNA sequence. Supershift analysis revealed that the gel-shifted complexes were comprised of p65 (RelA) and p50 (NF-kappaB1). Northern blot analyses demonstrated both the induction of vascular cell adhesion molecule 1 and E-selectin and the upregulation of intercellular adhesion molecule 1 mRNA in HUVEC infected with T. cruzi. Immunocytochemical staining confirmed adhesion molecule expression in response to T. cruzi infection. These findings are consistent with the hypothesis that the activation of the NF-kappaB pathway in endothelial cells associated with T. cruzi infection may be an important factor in the inflammatory response and subsequent vascular injury and endothelial dysfunction that lead to chronic cardiomyopathy. PMID: 10496926 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 199: Am J Physiol. 1999 Sep;277(3 Pt 1):L523-32. Endotoxin-specific NF-kappaB activation in pulmonary epithelial cells harboring adenovirus E1A. Keicho N, Higashimoto Y, Bondy GP, Elliott WM, Hogg JC, Hayashi S. Third Department of Internal Medicine, University of Tokyo, Tokyo 113, Japan. Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-kappaB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-kappaB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c-jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-kappaB induction in these cells. PMID: 10484459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 200: FEMS Microbiol Lett. 1999 Sep 1;178(1):117-22. Listeria monocytogenes infection of Caco-2 human epithelial cells induces activation of transcription factor NF-kappa B/Rel-like DNA binding activities. Hauf N, Goebel W, Fiedler F, Kuhn M. Lehrstuhl fur Mikrobiologie, Theodor-Boveri-Institut fur Biowissenschaften der Universitat Wurzburg, Germany. kuhn@biozentrum.uni-wuerzburg.de The effect of Listeria monocytogenes infection on the cellular level of transcription factor NF-kappa B in the human epithelia-like cell line Caco-2 was investigated. Infection with L. monocytogenes or treatment with lipoteichoic acid induced the formation of three NF-kappa B-like DNA-protein complexes C1, C2, and C3, which were identified as containing either, RelA and p50, RelB and p50, or p50, respectively. NF-kappa B activation in L. monocytogenes-infected Caco-2 cells was distinct from NF-kappa B activation in infected P388D1 macrophages concerning the NF-kappa B complexes formed and the kinetics of the induction. PMID: 10483730 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 201: Blood. 1999 Sep 15;94(6):1878-89. Nuclear factor-kappaB-dependent induction of interleukin-8 gene expression by tumor necrosis factor alpha: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation. Vlahopoulos S, Boldogh I, Casola A, Brasier AR. Department of Internal Medicine, Sealy Center for Molecular Sciences, Galveston, TX, USA. Tumor necrosis factor alpha (TNFalpha) is a pluripotent activator of inflammation by inducing a proinflammatory cytokine cascade. This phenomenon is mediated, in part, through inducible expression of the CXC chemokine, interleukin-8 (IL-8). In this study, we investigate the role of TNFalpha-inducible reactive oxygen species (ROS) in IL-8 expression by "monocyte-like" U937 histiocytic lymphoma cells. TNFalpha is a rapid activator of IL-8 gene expression by U937, producing a 50-fold induction of mRNA within 1 hour of treatment. In gene transfection assays, the effect of TNFalpha requires the presence of an inducible nuclear factor-kappaB (NF-kappaB) (Rel A) binding site in the IL-8 promoter. TNFalpha treatment induces a rapid translocation of the 65 kD transcriptional activator NF-kappaB subunit, Rel A, whose binding in the nucleus occurs before changes in intracellular ROS. Pretreatment (or up to 15 minutes posttreatment) relative to TNFalpha with the antioxidant dimethyl sulfoxide (DMSO) (2% [vol/vol]) blocks 80% of NF-kappaB-dependent transcription. Surprisingly, however, DMSO has no effect on inducible Rel A binding. Similar selective effects on NF-kappaB transcription are seen with the unrelated antioxidants, N-acetylcysteine (NAC) and vitamin C. These data indicate that TNFalpha induces a delayed ROS-dependent signalling pathway that is required for NF-kappaB transcriptional activation and is separable from that required for its nuclear translocation. Further definition of this pathway will yield new insights into inflammation initiated by TNFalpha signalling. PMID: 10477716 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 202: J Biol Chem. 1999 Sep 10;274(37):26448-53. Inhibition of interleukin-1-stimulated NF-kappaB RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity. Egan LJ, Mays DC, Huntoon CJ, Bell MP, Pike MG, Sandborn WJ, Lipsky JJ, McKean DJ. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905, USA. egan.laurence@mayo.edu Nuclear factor kappaB (NF-kappaB) is an inducible transcription factor that regulates genes important in immunity and inflammation. The activity of NF-kappaB is highly regulated: transcriptionally active NF-kappaB proteins are sequestered in the cytoplasm by inhibitory proteins, IkappaB. A variety of extracellular signals, including interleukin-1 (IL-1), activate NF-kappaB by inducing phosphorylation and degradation of IkappaB, allowing nuclear translocation and DNA binding of NF-kappaB. Many of the stimuli that activate NF-kappaB by inducing IkappaB degradation also cause phosphorylation of the NF-kappaB RelA (p65) polypeptide. The transactivating capacity of RelA is positively regulated by phosphorylation, suggesting that in addition to cytosolic sequestration by IkappaB, phosphorylation represents another mechanism for control of NF-kappaB activity. In this report, we demonstrate that mesalamine, an anti-inflammatory aminosalicylate, dose-dependently inhibits IL-1-stimulated NF-kappaB-dependent transcription without preventing IkappaB degradation or nuclear translocation and DNA binding of the transcriptionally active NF-kappaB proteins, RelA, c-Rel, or RelB. Mesalamine was found to inhibit IL-1-stimulated RelA phosphorylation. These data suggest that pharmacologic modulation of the phosphorylation status of RelA regulates the transcriptional activity of NF-kappaB, independent of nuclear translocation and DNA binding. These findings highlight the importance of inducible phosphorylation of RelA in the control of NF-kappaB activity. PMID: 10473604 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 203: Cell Growth Differ. 1999 Aug;10(8):537-44. The inhibitory effects of transforming growth factor beta1 on breast cancer cell proliferation are mediated through regulation of aberrant nuclear factor-kappaB/Rel expression. Sovak MA, Arsura M, Zanieski G, Kavanagh KT, Sonenshein GE. Department of Pathology and Laboratory Medicine, Boston University Medical School, Massachusetts 02118, USA. Nuclear factor (NF)-kappaB/Rel transcription factors normally exist in non-B cells, such as epithelial cells, in inactive forms sequestered in the cytoplasm with specific inhibitory proteins, termed IkappaBs. Recently, however, we discovered that breast cancer is typified by aberrant constitutive expression of NF-kappaB/Rel factors. Because these factors control genes that regulate cell proliferation, here we analyzed the potential role of NF-kappaB/Rel in the ability of transforming growth factor (TGF)-beta1 to inhibit the growth of breast cancer cells. The decreased growth of Hs578T and MCF7 breast cancer cell lines on TGF-beta1 treatment correlated with a drop in NF-kappaB/Rel binding. This decrease was due to the stabilization of the inhibitory protein IKB-alpha. Ectopic expression of c-Rel in Hs578T cells led to the maintenance of NF-kappaB/Rel binding and resistance to TGF-beta1-mediated inhibition of proliferation. Similarly, expression of the p65 subunit ablated the inhibition of Hs578T cell growth mediated by TGF-beta1. Thus, the inhibition of the aberrantly activated, constitutive NF-kappaB/Rel plays an important role in the arrest of the proliferation of breast cancer cells, which suggests that NF-kappaB/Rel may be a useful target in the treatment of breast cancer. PMID: 10470853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 204: Oncogene. 1999 Aug 12;18(32):4554-63. Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA. Wang W, Abbruzzese JL, Evans DB, Chiao PJ. Department of Surgical Oncology, The University of Texas MD Andersen Cancer Center, Houston, Texas, TX 77030, USA. The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential. PMID: 10467400 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 205: Mol Cell Biol. 1999 Sep;19(9):6140-53. Identification by in vivo genomic footprinting of a transcriptional switch containing NF-kappaB and Sp1 that regulates the IkappaBalpha promoter. Algarte M, Kwon H, Genin P, Hiscott J. Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and Departments of Microbiology & Immunology, Medicine, and Oncology, McGill University, Montreal, Canada H3T 1E2. In unstimulated cells, NF-kappaB transcription factors are retained in the cytoplasm by inhibitory IkappaB proteins. Upon stimulation by multiple inducers including cytokines or viruses, IkappaBalpha is rapidly phosphorylated and degraded, resulting in the release of NF-kappaB and the subsequent increase in NF-kappaB-regulated gene expression. IkappaBalpha gene expression is also regulated by an NF-kappaB autoregulatory mechanism, via NF-kappaB binding sites in the IkappaBalpha promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) progressively decreased endogenous IkappaBalpha protein levels. In the present study, we demonstrate that expression of TD-IkappaBalpha blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced IkappaBalpha gene transcription and abolished NF-kappaB DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IkappaBalpha. In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the -63 to -53 kappaB1 site but also to the adjacent -44 to -36 Sp1 site of the IkappaBalpha promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-IkappaBalpha expression. Prolonged NF-kappaB binding and a temporal switch in the composition of NF-kappaB complexes bound to the -63 to -53 kappaB1 site of the IkappaBalpha promoter were also observed; with time after induction, decreased levels of transcriptionally active p50-p65 and increased p50-c-Rel heterodimers were detected at the kappaB1 site. Mutation of either the kappaB1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the IkappaBalpha promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-kappaB and Sp1 that is essential for autoregulation of the IkappaBalpha promoter. PMID: 10454561 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 206: Autoimmunity. 1999;30(2):115-28. Aberrant expression of the NF-kappaB and IkappaB proteins in B cells from viable motheaten mice. Khaled AR, Butfiloski EJ, Villas B, Sobel ES, Schiffenbauer J. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville 32610, USA. In viable motheaten mice, a mutation in the gene encoding the phosphatase, SHP1, causes severe immunodeficiency and autoimmunity. A defective phosphatase may result in modified phosphorylation of proteins involved in gene regulation. Since the NFkappaB/IkappaB proteins are regulated through phosphorylation, we wished to understand if the expression of these proteins was altered by the SHP1 defect. Splenic B cells from viable motheaten mice were isolated and assessed for purity by flow cytometry. Levels of each protein in isolated B cells were examined by Western blot analyses. Measurement of RNA levels for each protein was assessed by semi-quantitative RT-PCR. Western blots revealed that, in me(v) whole cell lysates, there were reduced levels of RelA and RelB proteins and increased levels of p50 and c-Rel. Furthermore, we analyzed the protein levels of IkappaBalpha and found that, in me(v), this inhibitor was significantly reduced, while the level of another member of the IkappaB family, IkappaBbeta, was not. To determine if these findings in me(v) were secondary to the autoimmune process, we evaluated NF-kappaB/IkappaB expression in the BXSB murine model of autoimmunity. Unlike me(v), B cells from BXSB/Yaa mice had NF-kappaB complexes composed of the RelA submit, and IkappaBalpha was readily detected. In addition, RNA for the RelA and IkappaBalpha proteins in me(v) and control littermates was detected by RT-PCR, indicating that the reduced amounts of these proteins was not exclusively due to transcriptional defects. We conclude that the differences in NF-kappaB/IkappaB proteins that we have described in me(v) are likely a consequences of the SHP1 defect and could contribute to the clinical disorder that characterizes me(v) mice. PMID: 10435725 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 207: Dev Genet. 1999;25(1):23-30. Trophoblast giant cells express NF-kappa B2 during early mouse development. Muggia A, Teesalu T, Neri A, Blasi F, Talarico D. Dipartimento di Genetica e Biologia dei Microrganismi dell'Universita di Milano, Italy. To investigate whether transcription factors of the NF-kappa B family could play a role in early mammalian development, we have analyzed the expression of nfkb1, nfkb2, c-Rel, RelA, RelB, and bcl-3 from 6.5- to 10.5-day mouse embryo implantation sites. Our study shows that nfkb2 mRNA and protein are specifically localized in trophoblast giant cells throughout the stages analyzed. Trophoblast giant cells obtained upon in vitro cultures of 7.5-day ectoplacental cones display NF-kappa B DNA-binding activity that is supershifted by the anti-NF-kappa B2 antibody. Trophoblast giant cells are embryo-derived cells that form an interface between embryonic and maternal tissues during early mouse development; they are involved in decidual remodeling and expansion of the embryonic cavity, placenta formation, and possibly avoidance of maternal immune response to the embryo. Our study suggests that NF-kappa B2 could play a role in the modulation of genes expressed in trophoblast giant cells during the course of early embryogenesis, and therefore be relevant for tissue remodeling and morphogenesis of placenta. PMID: 10402669 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 208: J Korean Med Sci. 1999 Jun;14(3):299-303. Suppression of NF-kappaB activation in normal T cells by supernatant fluid from human renal cell carcinomas. Kim HJ, Park JK, Kim YG. Department of Urology, Institute for Medical Science, Chonbuk National University Medical School, Chonju, Korea. hjkim@moak.chonbuk.ac.kr T lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined whether supernatant fluid from RCC explants (RCC-S) induced the same phenotype of NF-kappaB suppression in normal T cells that is observed in patient T cells. The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, no activation of RelA/NFkappaB1-binding activity was detectable, while in others kappaB-binding activity was modestly induced but the duration was reduced. IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did not alter the cytoplasmic levels of RelA and NF-kappaB1 but did suppress their nuclear localization and inhibited the activation of RelA/NF-kappaB1 binding complexes. These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also appears that NF-kappaB suppression by RCC-S may contribute to the immunosuppression of host immunity. PMID: 10402173 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 209: Toxicol Appl Pharmacol. 1999 Jun 15;157(3):213-21. Involvement of nuclear factor-kappaB in a murine model for the acute form of autoimmune-like toxic oil syndrome. Bell SA, Page S, Baumgartner B, Berking C, Haas M, Eisele T, Neumeier D, Brand K. Department of Dermatology, Ludwig-Maximilians-University, Munich, 80337, Germany. The toxic oil syndrome (TOS) represents an exogenously induced autoimmune disease with acute or chronic symptoms similar to systemic lupus erythematosus or scleroderma. When genetically different mouse strains were exposed to oleic acid anilide (OAA), it was possible to mimic the different syndrome manifestations. The aim of the present study was to examine the role of NF-kappaB/Rel transcription factors in the development of the severe acute wasting disease observed in A/J mice. Within a week of OAA exposure, the A/J, but not B10.S strain, displayed weight loss, cachexia, apathy, reduced activity, and breathing difficulties. In affected A/J mice we observed a marked increase in NF-kappaB activation (p50/p65 dimers) both in splenic T cells and peritoneal macrophages as well as in tissue from aorta and gut. Incubation of splenocytes with OAA in vitro induced a dose-dependent removal of IkappaB-alpha, accompanied by NF-kappaB activation, whereas Sp-1 binding was not affected. Furthermore, we demonstrated the increased expression of the two NF-kappaB target genes IL-6 and IL-1beta in OAA-exposed mice and a transient OAA-induced accumulation of TNFalpha in vitro. This is the first report which implicates NF-kappaB/Rel in acute forms of chemically induced autoimmune-like disease and may serve as a paradigm for the involvement of this transcriptional system in acute processes associated with autoimmunity, suggesting possible avenues of therapeutic intervention. Copyright 1999 Academic Press. PMID: 10373405 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 210: Radiat Res. 1999 Jun;151(6):703-9. A high dose of ionizing radiation induces tissue-specific activation of nuclear factor-kappaB in vivo. Zhou D, Brown SA, Yu T, Chen G, Barve S, Kang BC, Thompson JS. Department of Internal Medicine, University of Kentucky, Lexington 40536, USA. Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) is one of the important responses of cells to an external stress such as ionizing radiation. We studied radiation-induced NF-kappaB activation in vivo in male BALB/c mice. After the mice were exposed to 8.5 Gy total-body gamma irradiation, the spleen, mesenteric lymph nodes, thymus, liver, lung, colon, brain and bone marrow were harvested 1, 2.5, 5, 10 and 20 h postirradiation. NF-kappaB DNA-binding activity was analyzed in the nuclear protein extracts by a gel shift assay. When compared to the levels in untreated control mice, radiation induced activation of NF-kappaB in spleen, mesenteric lymph nodes and bone marrow but not in the other tissues examined. In contrast, an i.p. injection of a lethal dose (3 mg/kg) of lipopolysaccharide also increased activity of NF-kappaB in the liver and lung. The gel supershift assay with Nfkb1, Rela and/or Rel antibodies revealed that the specific molecular forms of NF-kappaB activated by radiation in the spleen were Nfkb1 homodimers and Nfkb1/Rela heterodimers. In mesenteric lymph nodes, the heterodimerized Rel/Rela NF-kappaB was also activated. In bone marrow, an NF-kappaB-like binding factor was induced that may be Nfkb1/Rela- and Rel/Rela-like heterodimers, but it exhibited a higher mobility than Nfkb1 homodimers. These results indicate that in vivo, ionizing radiation induces NF-kappaB activation that varies in both tissue distribution and moleoular composition. PMID: 10360790 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 211: Oncogene. 1999 Apr 22;18(16):2663-6. INK4 cell cycle inhibitors direct transcriptional inactivation of NF-kappaB. Wolff B, Naumann M. Max-Planck-Institut fur Infektionsbiologie, Abteilung Molekulare Biologie, Berlin, Germany. The nuclear factor kappaB, a transcription factor regulating the expression of multiple genes including genes essential for cell cycle control, is found in most cells in a dormant state in the cytoplasm bound to the inhibitory family I kappaB via an ankyrin repeat domain. Stimulation of cells with a variety of inducers inactivates I kappaB proteins. The active dimeric NF-kappaB complex, often composed of 50- and 65-kilodalton subunits of the Rel family, translocates into the nucleus, where the NF-kappaBp65 subunit stimulates transcription. Here we report that a family of proteins containing ankyrin repeats, the inhibitors of Cdk4 (INK4) is able to bind NF-kappaBp65. The association of p16INK4 with NF-kappaBp65 is considerable in HeLa- or 293 cells, if the NF-kappaB inhibitor I kappaB alpha is degraded in response to TNFalpha stimulation. Overexpression of INK4 molecules suppresses the transactivational ability of NF-kappaB significantly. In contrast to INK4 proteins, the cell cycle inhibitor p27 enhances NF-kappaB transactivation activity. Thus, the effect of INK4 proteins on NF-kappaB function possibly modifies NF-kappaB mediated transcriptional activation of cell cycle associated factors. PMID: 10353611 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 212: J Immunol. 1999 Jun 1;162(11):6442-50. In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. Ferreira V, Sidenius N, Tarantino N, Hubert P, Chatenoud L, Blasi F, Korner M. Laboratoire d'Immunologie Cellulaire et Tissulaire, Centre National de la Recherche Scientifique UMR 7627, Hopital de la Pitie-Salpetriere, Paris, France. To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression. PMID: 10352258 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 213: EMBO J. 1999 May 17;18(10):2803-11. Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. Bash J, Zong WX, Banga S, Rivera A, Ballard DW, Ron Y, Gelinas C. Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854-5638, USA. Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Here, we show that Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. Importantly, jagged1 transcripts were also upregulated by endogenous NF-kappaB activation and this effect was inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers led to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlated with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 was found to be highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system. PMID: 10329626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 214: Circ Res. 1999 May 14;84(9):1095-109. Nuclear factor-kappaB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Xuan YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qiu Y, Li JJ, Bolli R. Experimental Research Laboratory, Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute, Louisville, KY, USA. Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-kappaB (NF-kappaB) and to elucidate the mechanisms that control the activation of NF-kappaB after an ischemic stimulus in vivo. A total of 152 chronically instrumented, conscious rabbits were used. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles, which elicits late PC, induced rapid activation of NF-kappaB, as evidenced by a marked increase in p65 content (+164%; Western immunoblotting) and NF-kappaB DNA binding activity (+306%; electrophoretic mobility shift assay) in nuclear extracts isolated 30 minutes after the last reperfusion. These changes were attenuated 2 hours after ischemic PC and resolved by 4 hours. Competition and supershift assays confirmed the specificity of the NF-kappaB DNA complex signals. The mobility of the NF-kappaB DNA complex was shifted by anti-p65 and anti-p50 antibodies but not by anti-c-Rel antibodies, indicating that the subunits of NF-kappaB involved in gene activation after ischemic PC consist of p65-p50 heterodimers. Pretreatment with the NF-kappaB inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15 minutes before ischemic PC) completely blocked the nuclear translocation and increased DNA binding activity of NF-kappaB. The same dose of DDTC completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that NF-kappaB activation is essential for the development of this phenomenon in vivo. The ischemic PC-induced activation of NF-kappaB was also blocked by pretreatment with Nomega-nitro-L-arginine (L-NA), a nitric oxide synthase (NOS) inhibitor, N-2-mercaptopropionyl glycine (MPG), a reactive oxygen species (ROS) scavenger, chelerythrine, a protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor (all given at doses previously shown to block late PC), indicating that ischemic PC activates NF-kappaB via formation of NO and ROS and activation of PKC- and tyrosine kinase-dependent signaling pathways. A subcellular redistribution and increased DNA binding activity of NF-kappaB quantitatively similar to those induced by ischemic PC could be reproduced pharmacologically by giving the NO donor diethylenetriamine/NO (DETA/NO) (at a dose previously shown to elicit late PC), demonstrating that NO in itself can activate NF-kappaB in the heart. Taken together, these results provide direct evidence that activation of NF-kappaB is a critical step in the signal transduction pathway that underlies the development of the late phase of ischemic PC in conscious rabbits. The finding that four different pharmacological manipulations (L-NA, MPG, chelerythrine, and lavendustin A) produced similar inhibition of NF-kappaB suggests that this transcription factor is a common downstream pathway through which multiple signals elicited by ischemic stress (NO, ROS, PKC, tyrosine kinases) act to induce gene expression. To our knowledge, this is the first demonstration that NO can promote NF-kappaB activation in the heart, a finding that identifies a new biological function of NO and may have important implications for various pathophysiological conditions in which NO is involved and for nitrate therapy. PMID: 10325247 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 215: J Immunol. 1999 May 15;162(10):5853-9. Fibroblast growth factor-1 (FGF-1) enhances IL-2 production and nuclear translocation of NF-kappaB in FGF receptor-bearing Jurkat T cells. Byrd VM, Ballard DW, Miller GG, Thomas JW. Departments of Medicine and Microbiology/Immunology and Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. victor.m.byrd@vanderbilt.edu Fibroblast growth factors (FGFs) are heparin-binding proteins crucial to embryogenesis, angiogenesis, and wound healing. FGF-1 is abundantly expressed in the synovium in rheumatoid arthritis and in rejecting allografts, sites of chronic immune-mediated inflammation. The frequency of FGF-1-responsive T cells is increased in the peripheral blood of these disorders, and a high percentage of infiltrating T cells in rheumatoid arthritis synovium express receptors for FGF-1. To understand the action of FGF-1 in T cells, studies were initiated in Jurkat T cells that express the signaling isoform of FGF receptor-1. These experiments show that FGF-1 stimulation of Jurkat T cells provides a second signal that augments TCR-mediated IL-2 production. Analogous to costimulation via CD28, this activity is mediated through activation of Rel/kappaB, a family of transcription factors known to regulate IL-2 and other activation-inducible proteins. FGF-1 alone induces modest nuclear translocation of kappaB-binding proteins, and this translocation is enhanced by the combination of anti-CD3 and FGF-1. This NF-kappaB binding complex is composed of transcriptionally active p65(RelA)/p50 heterodimers and results primarily from the targeted degradation of IkappaB-alpha, an inhibitor that sequesters Rel/kappaB in the cytoplasm. These data are the first to show a connection between FGF-1 signaling and NF-kappaB activation outside of embryonic development. The signaling events that link FGF receptor-1 engagement and NF-kappaB activation in Jurkat are probably distinct from the CD28 costimulation pathway, since FGF-1-induced Rel/kappaB binding proteins do not contain significant levels of c-Rel and are not identical with the CD28 response complex. PMID: 10229820 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 216: J Immunol. 1999 May 1;162(9):5466-76. Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. Rahman A, Anwar KN, True AL, Malik AB. Department of Pharmacology, College of Medicine, University of Illinois, Chicago 60612, USA. ARahman@uic.edu We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation. PMID: 10228026 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 217: J Biol Chem. 1999 May 7;274(19):13594-603. Regulation of NF-kappaB RelA phosphorylation and transcriptional activity by p21(ras) and protein kinase Czeta in primary endothelial cells. Anrather J, Csizmadia V, Soares MP, Winkler H. Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. janarthe@caregrouyp.harvard.edu The activity of the transcription factor NF-kappaB is thought to be regulated mainly through cytoplasmic retention by IkappaB molecules. Here we present evidence of a second mechanism of regulation acting on NF-kappaB after release from IkappaB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-kappaB through a pathway involving activation of protein kinase Czeta (PKCzeta) and p21(ras). We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCzeta or p21(ras) results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation. PMID: 10224130 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 218: Brain Res Mol Brain Res. 1999 Apr 20;67(2):303-15. Characterization of a neuronal kappaB-binding factor distinct from NF-kappaB. Moerman AM, Mao X, Lucas MM, Barger SW. Department of Geriatrics, University of Arkansas for Medical Sciences, J.L. McClellan Memorial Veterans Affairs Medical Center, 4300 W. Seventh St., Little Rock, AR 72205, USA. Transcription factors that bind kappaB enhancer elements have begun to garner wide attention in neurobiology. Data suggest that activation of kappaB-binding factors in neurons can be protective against various neurotoxins, but other data have connected NF-kappaB to cell death. In electrophoretic mobility shift assays of kappaB-binding activity, we have found that the predominant activity in rat brain tissue, in primary neurons, and in neuronal cell lines has a mobility inconsistent with that of bona fide NF-kappaB (RelA-p50 heterodimer). We have tentatively termed this activity neuronal kappaB-binding factor (NKBF). Competition assays with various DNA probes distinguished NKBF from NF-kappaB. Probes that efficiently bind the p50 homodimer were able to compete with a conventional NF-kappaB probe for NKBF binding, but NKBF did not react with antibodies to p50 (or any other known Rel family members). Furthermore, UV-crosslinking indicated that NKBF is composed of two polypeptides of 82 kDa and 27 kDa. Although NKBF activity can be elevated in a manner independent of new macromolecular synthesis, it does not appear to be modulated by IkappaB. Finally, no NF-kappaB was induced by glutamate in highly enriched neuronal cultures, although it was induced in neuron-glia cocultures. These data suggest that the primary kappaB-binding transcription factor in neurons is a novel protein complex distinct from NF-kappaB. Copyright 1999 Elsevier Science B.V. PMID: 10216229 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 219: Circ Res. 1999 Apr 2;84(6):695-703. Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-kappa B transcription factors. Han Y, Runge MS, Brasier AR. Department of Internal Medicine, University of Texas Medical Branch, Galveston, Tex.77555-1060, USA. Interleukin-6 (IL-6) is a multifunctional cytokine expressed by angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs) that functions as an autocrine growth factor. In this study, we analyze the mechanism for Ang II-inducible IL-6 expression in quiescent rat VSMCs. Stimulation with the Ang II agonist Sar1 Ang II (100 nmol/L) induced transcriptional expression of IL-6 mRNA transcripts of 1.8 and 2.4 kb. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, Sar1 Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-kappaB (NF-kappaB) motif. Sar1 Ang II induced cytoplasmic-to-nuclear translocation of the NF-kappaB subunits Rel A and NF-kappaB1 with parallel changes in DNA-binding activity in a biphasic manner, which produced an early peak at 15 minutes followed by a nadir 1 to 6 hours later and a later peak at 24 hours. The early phase of NF-kappaB translocation was dependent on weak simultaneous proteolysis of the IkappaBalpha and beta inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-kappaB1 form. Pretreatment with a potent inhibitor of IkappaBalpha proteolysis, TPCK, completely blocked Sar1 Ang IIAng II-induced NF-kappaB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-kappaB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-kappaB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs. PMID: 10189357 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 220: J Biol Chem. 1999 Apr 9;274(15):10145-53. The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis. Liou ML, Liou HC. Division of Immunology, Department of Medicine, Cornell University Medical College, New York, New York 10021, USA. hcliou@mail.med.cornell.edu The tumor necrosis factor receptor, p60 (TNF-R1), transduces death signals via the association of its cytoplasmic domain with several intracellular proteins. By screening a mammalian cDNA library using the yeast two-hybrid cloning technique, we isolated a ubiquitin-homology protein, DAP-1, which specifically interacts with the cytoplasmic death domain of TNF-R1. Sequence analysis reveals that DAP-1 shares striking sequence homology with the yeast SMT3 protein that is essential for the maintenance of chromosome integrity during mitosis (Meluh, P. B., and Koshland, D. (1995) Mol. Biol. Cell 6, 793-807). DAP-1 is nearly identical to PIC1, a protein that interacts with the PML tumor suppressor implicated in acute promyelocytic leukemia (Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P. S. (1996) Oncogene 13, 971-982), and the sentrin protein, which associates with the Fas death receptor (Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., and Yeh, E. T. (1996) J. Immunol. 157, 4277-4281). The in vivo interaction between DAP-1 and TNF-R1 was further confirmed in mammalian cells. In transient transfection assays, overexpression of DAP-1 suppresses NF-kappaB/Rel activity in 293T cells, a human kidney embryonic carcinoma cell line. Overexpression of either DAP-1 or sentrin causes apoptosis of TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant osteosarcoma cell line, U2OS. Furthermore, the dominant negative Fas-associated death domain protein (FADD) protein blocks the cell death induced by either DAP-1 or FADD. Collectively, these observations highly suggest a role for DAP-1 in mediating TNF-induced cell death signaling pathways, presumably through the recruitment of FADD death effector. PMID: 10187798 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 221: Blood. 1999 Apr 1;93(7):2360-8. Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells. Mori N, Fujii M, Ikeda S, Yamada Y, Tomonaga M, Ballard DW, Yamamoto N. Department of Preventive Medicine and AIDS Research, Research Field of Pathogenesis and Clinical Sciences, Institute of Tropical Medicine, Nagasaki University, Japan. Human T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-kappaB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-kappaB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I-infected T-cell lines, display constitutive NF-kappaB DNA binding activity and increased degradation of IkappaBalpha (an inhibitor of NF-kappaB). Whereas the NF-kappaB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-kappaB activity, as well as enhanced degradation of IkappaBalpha, despite the lack of detectable Tax expression. Interestingly, the NF-kappaB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-kappaB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-kappaB subunit activation. PMID: 10090947 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 222: J Biol Chem. 1999 Mar 26;274(13):8708-16. Overexpression of RelA causes G1 arrest and apoptosis in a pro-B cell line. Sheehy AM, Schlissel MS. Graduate Program in Immunology, Departments of Medicine, Molecular Biology & Genetics, and Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. NF-kappaB/Rel family proteins form a network of post-translationally regulated transcription factors that respond to a variety of extracellular stimuli and mediate distinct cellular responses. These responses include cytokine gene expression, regulated cell cycle activation, and both the protection from and induction of the cell death program. To examine the function of individual Rel family proteins in B cell development and resolve their role in the signaling of apoptosis, we used a tetracycline-regulated gene expression system to overexpress either c-Rel or RelA in the transformed pro-B cell line 220-8. Elevated levels of RelA, but not c-Rel, induced a G1 cell cycle arrest followed by apoptosis. Both the DNA binding and transactivation domains of RelA were required for this effect. When RelA was overexpressed in the immature B cell line WEHI 231 or the mature B cell line M12, neither cell cycle arrest nor apoptosis was evident. The differential effects of elevated RelA levels in these cell lines suggests that susceptibility to NF-kappaB-induced apoptosis may reflect a relevant selection event during B cell development. PMID: 10085110 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 223: Oncogene. 1999 Jan 28;18(4):943-53. Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells. Krappmann D, Emmerich F, Kordes U, Scharschmidt E, Dorken B, Scheidereit C. Max-Delbruck-Centrum for Molecular Medicine, Berlin, Germany. A common characteristic of malignant cells derived from patients with Hodgkin's disease (HD) is a high level of constitutive nuclear NF-kappaB/Rel activity, which stimulates proliferation and confers resistance to apoptosis. We have analysed the mechanisms that account for NF-kappaB activation in a panel of Hodgkin/Reed-Sternberg (H-RS) cell lines. Whereas two cell lines (L428 and KMH-2) expressed inactive IkappaBalpha, no significant changes in NF-kappaB or IkappaB expression were seen in other H-RS cells (L591, L1236 and HDLM-2). Constitutive NF-kappaB was susceptible to inhibition by recombinant IkappaBalpha, suggesting that neither mutations in the NF-kappaB genes nor posttranslational modifications of NF-kappaB were involved. Endogenous IkappaBalpha was bound to p65 and displayed a very short half-life. IkappaBalpha degradation could be blocked by inhibitors of the NF-kappaB activating pathway. Proteasomal inhibition caused an accumulation of phosphorylated IkappaBalpha and a reduction of NF-kappaB activity in HDLM-2 and L1236 cells. By in vitro kinase assays we demonstrate constitutive IkappaB kinase (IKK) activity in H-RS cells, indicating ongoing signal transduction. Furthermore, H-RS cells secrete one or more factor(s) that were able to trigger NF-kappaB activation. We conclude that aberrant activation of IKK's, and in some cases defective IkappaBs, lead to constitutive nuclear NF-kappaB activity, which in turn results in a growth advantage of Hodgkin's disease tumor cells. PMID: 10023670 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 224: J Immunol. 1999 Feb 15;162(4):1941-6. The p65 subunit of NF-kappa B is redundant with p50 during B cell proliferative responses, and is required for germline CH transcription and class switching to IgG3. Horwitz BH, Zelazowski P, Shen Y, Wolcott KM, Scott ML, Baltimore D, Snapper CM. Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA. B cells lacking individual NF-kappa B/Rel family members exhibit defects in activation programs. We generated small resting B cells lacking p65 or p50 alone, or lacking both p50 and p65, then evaluated the ability of these cells to proliferate, secrete Ig, and undergo Ig class switching. B cells lacking p65 proliferated well in response to all stimuli tested. However, these cells demonstrated an isolated defect in switching to IgG3, which was associated with a decrease in gamma 3 germline CH gene expression. Whereas, previously reported, B cells lacking p50 alone had a severe proliferative defect in response to LPS, a moderate defect in response to CD40 ligand (CD40L), and normal proliferation to Ag receptor cross-linking using dextran-conjugated anti-IgD Abs (alpha delta-dex), B cells lacking both p50 and p65 exhibited severely impaired proliferation in response to LPS, alpha delta-dex, and CD40L. This defect could be overcome by simultaneous administration of alpha delta-dex and CD40L. In response to this latter combination of stimuli, B cells lacking both p50 and p65 secreted Ig and underwent isotype switching to IgG1 as efficiently as B cells lacking p50 alone. These data demonstrate a role for the p65 subunit of NF-kappa B in germline CH gene expression as well as functional redundancy between p50 and p65 during proliferative responses. PMID: 9973462 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 225: Invest Ophthalmol Vis Sci. 1999 Feb;40(2):477-86. Suppression of NF-kappaB-dependent proinflammatory gene expression in human RPE cells by a proteasome inhibitor. Wang XC, Jobin C, Allen JB, Roberts WL, Jaffe GJ. Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina, USA. PURPOSE: To determine whether nuclear transcription factor-kappaB (NF-kappaB) is activated in human retinal pigment epithelial (hRPE) cells in response to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone or in combination and if so, whether expression of proinflammatory genes induced by these agents can be blocked by a proteasome inhibitor, MG-132, which inhibits the degradation of I kappaB, an NF-kappaB inhibitor, thereby preventing nuclear translocation of NF-kappaB. METHODS: Cultured hRPE were pretreated for 60 minutes with medium alone or medium containing the proteasome inhibitor MG-132 (20 microM) and then exposed to TNF-alpha (1.1 x 10(3) U/ml), IL-1beta (5 U/ml), or IFN-gamma (7.5 x 10(3) U/ml) alone or in combination (TII). Nuclear translocation of NF-kappaB was determined by fluorescence staining of the NF-kappaB Rel A (p65) subunit. Cytoplasmic I kappaB protein was measured by western blot analysis. Nuclear extract binding to kappaB DNA motifs was measured by electrophoretic mobility shift assay and antibody supershift assay. Steady state mRNA expression of the chemokines melanoma growth stimulating activity (MGSA)/gro-alpha, regulated on activation normal T-cell expression and secreted (RANTES), and monocyte chemoattractant protein (MCP-1), the cytokines IL-1beta and macrophage colony stimulating factor (M-CSF) and intercellular adhesion molecule-1 (ICAM-1) was evaluated by semiquantitative reverse transcription-polymerase chain reaction. Chemokine and cytokine protein secretion was measured by enzyme-linked immunosorbent assay. Cell-surface ICAM-1 expression was determined by flow cytometry. RESULTS: TNF-alpha, IL-1beta, and TII but not IFN-gamma alone caused degradation of I kappaB, Rel A nuclear translocation, and increased NF-kappaB DNA binding activity, effects that were blocked by pretreatment with MG-132. MG-132 suppressed MGSA/gro-alpha, RANTES, MCP-1, IL-1beta, M-CSF, and ICAM-1 mRNA expression and secreted RANTES, MCP-1, and M-CSF protein, and cell-surface ICAM-1 that were induced by IL-1beta, TNF-alpha, and TII. CONCLUSIONS: TNF-alpha, IL-1beta, and TII induce expression of proinflammatory cytokines and ICAM-1 in hRPE cells through an NF-kappaB-dependent signal transduction pathway. This effect is blocked by MG-132, a proteasome inhibitor that prevents I kappaB degradation. Inhibition of NF-kappaB may be a useful strategy to treat proliferative vitreoretinopathy and uveitis, ocular diseases initiated and perpetuated by cytokine activation. PMID: 9950608 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 226: Gastroenterology. 1999 Feb;116(2):420-30. Comment in: Gastroenterology. 1999 Feb;116(2):489-92. NF-kappaB/Rel activation in cerulein pancreatitis. Steinle AU, Weidenbach H, Wagner M, Adler G, Schmid RM. Department of Medicine I, University Ulm, Ulm, Germany. BACKGROUND & AIMS: Recent evidence suggests that a number of rapid signaling cascades are initiated during secretagogue-induced pancreatitis. However, little is known about the nuclear events. The aim of this study was to explore activation of the transcription factor NF-kappaB/Rel after supramaximal stimulation with the cholecystokinin analogue cerulein in the pancreas. METHODS & RESULTS: Nuclear appearance of NF-kappaB/Rel-binding activity was detectable 15 minutes after cerulein injection. The DNA-binding activity consisted of NF-kappaB1 p50, NF-kappaB2 p52, and RelA p65 as judged by supershift assays and Western blot analysis. The onset and termination of NF-kappaB/Rel activation correlated with the degradation and reappearance of IkappaBalpha. Cerulein in supramaximal but not in physiological doses activated NF-kappaB/Rel in vitro. After blocking of NF-kappaB/Rel activation with pyrrolidine dithiocarbamate, the degree of morphological alterations was more pronounced than in controls, serum amylase and lactate dehydrogenase levels were significantly increased, and messenger RNA levels of pancreatitis-associated protein were more strongly induced, reflecting a more severe degree of pancreatitis. Similar results were obtained when N-acetyl-L-cysteine was used as an inhibitor of NF-kappaB activation. CONCLUSIONS: These data show that NF-kappaB/Rel is rapidly activated during cerulein pancreatitis. This activation may induce a self-defending genetic program before the onset of cellular injury, which might prevent higher degrees of damage of pancreatic acinar cells after secretagogue hyperstimulation. PMID: 9922324 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 227: Clin Cancer Res. 1999 Jan;5(1):119-27. The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR, Chiao PJ. Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes. PMID: 9918209 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 228: Eur J Biochem. 1999 Jan;259(1-2):253-61. All three IkappaB isoforms and most Rel family members are stably associated with the IkappaB kinase 1/2 complex. Heilker R, Freuler F, Pulfer R, Di Padova F, Eder J. Novartis Pharma AG, Arthritis and Bone Metabolism Research, Basel, Switzerland. Nuclear factor kappa B (NF-kappaB) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappaB is present in the cytosol where it is complexed to its inhibitor IkappaB. Activation of NF-kappaB depends on the signal-induced phosphorylation of IkappaB by specific IkappaB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IkappaBalpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IkappaB kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1), the IkappaB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappaB-related and IkappaB-related proteins: RelA, RelB, cRel, p100, p105, Ikappa Balpha, Ikappa Bbeta and Ikappa Bepsilon. The existence of stable kinase-substrate complexes, the presence of all three known IkappaB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating Ikappa Balpha-, Ikappa Bbeta- and Ikappa Bepsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappaB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of Ikappa Balpha, we observed efficient phosphorylation of serine 32 in Ikappa Balpha by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation. PMID: 9914500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 229: Eur J Biochem. 1999 Jan;259(1-2):143-8. Rel transcription factors contribute to elevated urokinase expression in human ovarian carcinoma cells. Reuning U, Guerrini L, Nishiguchi T, Page S, Seibold H, Magdolen V, Graeff H, Schmitt M. Frauenklink der Technischen Universitat Munchen, Germany. ute.reuning@lrz.tu-muenchen.de Elevated levels of the urokinase-type plasminogen activator (uPA) in tumor cells are conductive to tumor cell spread and metastasis. In a previous study we observed that suppression of RelA dramatically reduced endogenous uPA synthesis in the human ovarian cancer cell line OV-MZ-6. Because the uPA promoter contains three potential Rel-like protein binding motifs (RRBE, 5'-NF-kappaB, and 3'-NF-kappaB) we conducted the first thorough systematic uPA promoter analysis to examine the direct impact of Rel proteins on uPA gene transcription. Disruption of RRBE resulted in a approximately 40% decrease in uPA promoter activity, mutation of the 5'-NF-kappaB motif led to an additional 20% decrease. The 3'-NF-kappaB motif was not active. Overexpression of RelA significantly enhanced uPA promoter activity, whereas IkappaB-alpha overexpression reduced uPA promoter activity by 40%. These data were supported by the finding that endogenous uPA was also increased sixfold by overexpression of RelA and decreased by 30% upon overexpression of IkappaB-alpha. Transfection of OV-MZ-6 cells with antisense deoxynucleotides directed to RelA expression reduced uPA promoter activity by at least 40%. Our data clearly suggest that by binding to uPA promoter elements, Rel transcripton factors contribute directly to elevated uPA gene expression in human ovarian cancer cells, thereby promoting the multiple functions of uPA during tumor growth and metastasis. PMID: 9914486 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 230: Cell. 1998 Dec 11;95(6):749-58. Comment in: Cell. 1998 Dec 11;95(6):729-31. Structure of an IkappaBalpha/NF-kappaB complex. Jacobs MD, Harrison SC. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. The inhibitory protein, IkappaBalpha, sequesters the transcription factor, NF-kappaB, as an inactive complex in the cytoplasm. The structure of the IkappaBalpha ankyrin repeat domain, bound to a partially truncated NF-kappaB heterodimer (p50/ p65), has been determined by X-ray crystallography at 2.7 A resolution. It shows a stack of six IkappaBalpha ankyrin repeats facing the C-terminal domains of the NF-kappaB Rel homology regions. Contacts occur in discontinuous patches, suggesting a combinatorial quality for ankyrin repeat specificity. The first two repeats cover an alpha helically ordered segment containing the p65 nuclear localization signal. The position of the sixth ankyrin repeat shows that full-length IkappaBalpha will occlude the NF-kappaB DNA-binding cleft. The orientation of IkappaBalpha in the complex places its N- and C-terminal regions in appropriate locations for their known regulatory functions. PMID: 9865693 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 231: J Neurosci. 1998 Dec 15;18(24):10356-65. Nerve growth factor-dependent activation of NF-kappaB contributes to survival of sympathetic neurons. Maggirwar SB, Sarmiere PD, Dewhurst S, Freeman RS. Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA. Neurotrophins activate multiple signaling pathways in neurons. However, the precise roles of these signaling molecules in cell survival are not well understood. In this report, we show that nerve growth factor (NGF) activates the transcription factors NF-kappaB and AP-1 in cultured sympathetic neurons. Activated NF-kappaB complexes were shown to consist of heterodimers of p50 and Rel proteins (RelA, as well as c-Rel), and NF-kappaB activation was found to occur independently of de novo protein synthesis but in a manner that required the action of the proteasome complex. Treatment with the NF-kappaB inhibitory peptide SN50 in the continuous presence of NGF resulted in dose-dependent induction of cell death. Under the conditions used, SN50 was shown to selectively inhibit NF-kappaB activation but not the activation of other cellular transcription factors such as AP-1 and cAMP response element-binding protein. Cells treated with SN50 exhibited morphological and biochemical hallmarks of apoptosis, and the kinetics of cell killing were accelerated relative to death induced by NGF withdrawal. Finally, experiments were conducted to test directly whether NF-kappaB could act as a survival factor for NGF-deprived neurons. Microinjection of cells with an expression plasmid encoding NF-kappaB (c-Rel) resulted in enhanced neuronal survival after withdrawal of NGF, whereas cells that were transfected with a vector encoding a mutated derivative of c-Rel lacking the transactivation domain underwent cell death to the same extent as control cells. Together, these findings suggest that the activation of NF-kappaB/Rel transcription factors may contribute to the survival of NGF-dependent sympathetic neurons. PMID: 9852573 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 232: Cell Death Differ. 1998 Nov;5(11):963-72. Rel blocks both anti-Fas- and TNF alpha-induced apoptosis and an intact Rel transactivation domain is essential for this effect. Zong WX, Bash J, Gelinas C. Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854-5638, USA. The v-Rel oncoprotein must be continuously expressed to prevent the apoptosis of transformed lymphoid cells, and also inhibits TNF alpha-induced cell death. A tetracycline-regulated cell system was used to characterize the functions necessary for the anti-apoptotic activity of Rel proteins. v-Rel mutants defective for DNA binding or transactivation showed no protective effect. Similarly, whereas the transcription-competent c-Rel and RelA proteins inhibited TNF alpha-induced cytolysis, the transactivation-negative p50/NF-kappa B1 did not. Importantly, this study is the first to show that c-Rel can also confer significant protection from Fas-mediated cell death. Since the TNFR1- and Fas-signaling pathways involve some intermediates that are common and others that are unique to each pathway, these findings indicate that c-Rel may regulate the expression of genes that function to antagonize either or both death-signaling pathways. PMID: 9846183 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 233: AIDS Res Hum Retroviruses. 1998 Nov 20;14(17):1509-19. Functional analysis of the proximal CCR5 promoter. Liu R, Zhao X, Gurney TA, Landau NR. Aaron Diamond AIDS Research Center, New York, New York 10016, USA. Two promoters for the CCR5 gene, termed Pu and Pd, corresponding to the upstream and downstream initiation sites, respectively, have been described. We show here that the proximal promoter, Pd, is used two- to fivefold more frequently than Pu in primary activated T cells and in the transformed T cell line PM1. Because of its importance in CCR5 transcription we characterized the transcriptional activity of this promoter. Pd contains a pair of consensus TATA elements (nt -19 and -31) and several potential regulatory elements and transcription factor-binding sites, including those for STAT, NF-kappaB, AP-1, NF-AT, and CD28RE. Using a transfected reporter vector, we found the promoter to be highly active and cell type specific. By 5' deletion analysis, the minimal CCR5 promoter was localized to a 225-nucleotide region (nt -189 to +36). This region contained the two TATA elements, a CD28RE consensus sequence, an AP-1-binding site, and two STAT-binding sites. The 1.9-kb intron appeared to have a negative influence on reporter gene activity, suggesting the presence of a negative element in this region. In addition, an upstream negative element was detected in the region nt -988 to -588. Mutagenesis of the TATA elements, of the NF-kappaB-, and AP-1-, and STAT-binding sites, and of the CD28RE indicated the importance of each of these in transcription. Finally, the NF-kappaB/Rel family member, p65(RelA), was a potent activator of the CCR5 promoter. PMID: 9840284 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 234: J Biol Chem. 1998 Dec 11;273(50):33561-5. Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappaB activity. Hu MC, Tang-Oxley Q, Qiu WR, Wang YP, Mihindukulasuriya KA, Afshar R, Tan TH. Department of Cell Biology, Amgen, Inc., Thousand Oaks, California 91320, USA. Nuclear factor kappaB (NF-kappaB) and the Rel family of proteins are pleiotropic transcription factors that play central roles in the immune and inflammatory responses, as well as apoptosis. Here, we identified a serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel, NF-kappaB p50, and RelA. The amino acid sequences of human and mouse PPX are 100% identical, and the PPX gene was mapped to human chromosome 16 p11.2. Overexpression of PPX, but not catalytically inactive PPX mutants, stimulated the DNA-binding activity of c-Rel and activated NF-kappaB-mediated transcription. These results suggest that PPX is a novel activator of c-Rel/NF-kappaB. PMID: 9837938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 235: Cell Growth Differ. 1998 Nov;9(11):949-59. Cooperative binding and synergistic activation by RelA and C/EBPbeta on the intercellular adhesion molecule-1 promoter. Catron KM, Brickwood JR, Shang C, Li Y, Shannon MF, Parks TP. Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA. kcatron@bi-pharm.com Intercellular adhesion molecule-1 (ICAM-1) is up-regulated on numerous cell types in response to inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha) activates the ICAM-1 promoter through a variant nuclear factor-kappaB (NF-kappaB) site at -187/-178 bp upstream of the transcription start site. In this investigation, we provide biochemical and functional evidence that an adjacent CCAAT/enhancer binding protein (C/EBP) site and this variant NF-kappaB site define a composite element for activation of the ICAM-1 promoter in certain cell lines. We detected an endogenous TNF-alpha-inducible DNA-protein complex in nuclear extracts from A549, HeLa, and EVC304 cells that contained both RelA and C/EBPbeta but not other family members. Complex formation required intact C/EBP and NF-kappaB sites and was absolutely dependent on translocation of RelA into the nucleus. Complex formation and cooperative binding were also demonstrated using recombinant proteins, and as above, both binding sites were necessary. Interestingly, the RelA/C/EBPbeta complex was not detected in either Jurkat or Raji cells, indicating cell type specificity. Functional studies with various reporter gene constructs revealed that both binding sites were required for maximal activation of the ICAM-1 promoter in response to TNF-alpha and for synergistic activation by RelA and C/EBPbeta. This is the first detailed analysis of how RelA and C/EBPbeta function to regulate ICAM-1 expression, and this study has important implications for how this gene is activated in specific cell types. PMID: 9831247 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 236: J Biol Chem. 1998 Dec 4;273(49):32460-6. Involvement of Jun and Rel proteins in up-regulation of interleukin-4 gene activity by the T cell accessory molecule CD28. Li-Weber M, Giasi M, Krammer PH. Tumor Immunology Program, German Cancer Research Center, D-69126 Heidelberg, Germany. m.li-weber@dkfz-heidelberg.de CD28 serves as a costimulatory cell surface molecule in T cell activation. CD28 signaling may also play a role in balancing the inflammatory/humoral (Th1/Th2) responses during an immune reaction. CD28 costimulation has been shown to promote the production of Th2 cytokines including interleukin (IL)-4, a key cytokine essential for Th2 differentiation and for the pathogenesis of allergic inflammation. In this study, we show that IL-4 mRNA and activity of the IL-4 promoter can be activated by the CD28 signal alone and are further augmented by CD28 costimulation of alpha-CD3- or mitogen-activated Jurkat T cells. Two important IL-4 enhancer elements, positive regulatory element (PRE)-I and P1, are found to respond to CD28 stimulation-induced transactivation. In contrast to the Th1 IL-2 CD28RE, activity of the IL-4 PRE-I and P1 can be induced by the CD28 signal alone. In correlation with CD28-induced transcriptional activation, AP-1 (c-Jun, JunD) and NF-kappaB/Rel (c-Rel, RelA) family members are found to bind to the two regulatory elements PRE-I and P1 upon CD28 stimulation. The data provide the first mapping of the CD28-responsive site in a Th2 cytokine gene, the IL-4 gene. They also show that the CD28 signal can directly activate a gene (e.g. IL-4) at the transcriptional level. PMID: 9829977 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 237: J Clin Pharmacol. 1998 Nov;38(11):981-93. Nuclear factor kappa B: important transcription factor and therapeutic target. Lee JI, Burckart GJ. Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh 15261, USA. Nuclear factor kappa B (NF-kappa B) is an ubiquitous rapid response transcription factor in cells involved in immune and inflammatory reactions, and exerts its effect by expressing cytokines, chemokines, cell adhesion molecules, growth factors, and immunoreceptors. In this manner, NF-kappa B contributes to immunologically mediated diseases such as allograft rejection, rheumatoid arthritis, and bronchial asthma. The prototypic inducible form of NF-kappa B is a heterodimer composed of NF-kB1 and RelA, which both belong to the NF-kappa B/Rel family of proteins. Inactive NF-kappa B is present in the cytoplasm complexed with an inhibitory protein, I kappa B. NF-kappa B is activated by a number of incoming signals from the cell surface. Released from I kappa B inhibition, NF-kappa B translocates into the nucleus and binds to the kappa B motif of the target gene. The NF-kappa B activation process can be inhibited by pharmacologic agents at each activation step. Glucocorticoids inhibit NF-kappa B by directly associating with NF-kappa B or by upregulating I kappa B expression. Cyclosporine and tacrolimus prevent NF-kappa B activation by inhibiting the action of calcineurin, a phosphatase that indirectly induces I kappa B degradation. Deoxyspergualin inhibits NF-kappa B by blocking its nuclear translocation. Aspirin and salicylates inhibit upstream events inducing I kappa B phosphorylation. Tepoxalin and antioxidants inhibit NF-kappa B activation by influencing the redox state of the cell. Further research is required to develop more specific inhibitors to treat diseases mediated by NF-kappa B. Publication Types: Review Review, Tutorial PMID: 9824778 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 238: J Leukoc Biol. 1998 Nov;64(5):667-74. Oxidized LDL modulates activation of NFkappaB in mononuclear phagocytes by altering the degradation if IkappaBs. Hamilton TA, Major JA, Armstrong D, Tebo JM. Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA. Oxidized low density lipoprotein (oxLDL) is known to alter the expression of inflammatory gene products in mononuclear phagocytes. The mechanisms involved in this effect were studied by examining the activation of nuclear factor kappaB (NFkappaB), a transcription factor known to be important in controlling the expression of such genes. Pretreatment of peritoneal macrophages with oxLDL modulated the activation of NFkappaB in response to either lipopolysaccharide (LPS) or the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). In macrophages pretreated with oxLDL the nuclear translocation of Rel family members (RelA and c-Rel) is delayed (LPS) or markedly diminished (IFN-gamma/IL-2) and results in delayed or reduced appearance of kappaB binding activity within the nucleus. These changes in NFkappaB activation result from alterations in the stimulus-dependent degradation of IkappaB alpha and IkappaB beta. The effects of oxLDL on NFkappaB activation depend both on the degree of LDL oxidation (most potent with extensive oxidation) and on the time of exposure of the cells to the lipoprotein preparation (a minimal exposure of 6 h is required before inhibitory effects are observed). The modulation of IkappaB/NFkappaB function in cells exposed to oxLDL appears to be responsible for previously reported effects of oxLDL on chemoattractant cytokine gene expression where both inhibition and delay of such stimulus-dependent events has been observed. PMID: 9823773 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 239: J Photochem Photobiol B. 1998 Aug 21;45(1):1-8. Nf-kappa B: an important transcription factor in photobiology. Legrand-Poels S, Schoonbroodt S, Matroule JY, Piette J. Laboratory of Virology, University of Liege, Belgium. Increased gene expression as a consequence of environmental stress is typically observed in mammalian cells. In the past few years the cis- and trans-acting genetic elements responsible for gene induction by radiation (from UV-C to visible light) started to be well characterized. The molecular mechanisms involved in the cell response to radiation reveal that an important control occurs at the transcriptional level and is coordinated by various transcription factors. Among these transcription factors, the well-known Rel/NF-kappa B family of vertebrate transcription factors plays a pivotal role as it controls both the inflammatory and immune responses. The NF-kappa B family comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or heterodimers. Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as I kappa B. In the best-characterized example, I kappa B-alpha interacts with a p50/RelA (NF-kappa B) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), I kappa B-alpha undergoes phosphorylation, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, while still complexed with NF- kappa B. Removal of I kappa B-alpha uncovers the nuclear localization signals on subunits of NF-kappa B, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. In this paper, we shall show that molecular mechanisms leading to NF-kappa B activation by UV or by photosensitization are initiated by oxidative damage at the membrane level or by the induction of DNA alterations. While the exact nature of the transduction intermediates is still unknown, we shall show that NF-kappa B activation by radiation follows different pathways from those used by pro-inflammatory cytokines. Publication Types: Review Review, Tutorial PMID: 9819895 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 240: Neuroreport. 1998 Sep 14;9(13):3029-32. Microglial activation in multiple system atrophy: a potential role for NF-kappaB/rel proteins. Schwarz SC, Seufferlein T, Liptay S, Schmid RM, Kasischke K, Foster OJ, Daniel S, Schwarz J. Department of Neurology, University of Ulm, Germany. Microglial activation is a prominent feature of affected brain areas in multiple system atrophy. Microglia express proinflammatory peptides, which may be a result of activation of nuclear factor-KB. We investigated the nuclear presence of RelA, the 65 kDa subunit of the NF-KB/RelA family in striatum and brain stem of patients with multiple system atrophy. Affected brain areas of patients with multiple system atrophy showed a marked immunoreactivity for nuclear Rel A p65, which was almost exclusively localized in activated microglia. Interestingly nuclear translocation of Rel A was not detected in striatal tissue of controls and Parkinson disease patients. Thus, NF-kappaB/Rel A complexes may play a role in mediating microglial activation in multiple system atrophy. PMID: 9804310 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 241: J Biol Chem. 1998 Nov 6;273(45):29411-6. Activation of nuclear factor-kappaB-dependent transcription by tumor necrosis factor-alpha is mediated through phosphorylation of RelA/p65 on serine 529. Wang D, Baldwin AS Jr. Lineberger Comprehensive Cancer Center and Department of Biology, CB 7295, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.p6 Nuclear factor-kappaB (NF-kappaB) is an essential transcription factor in the control of expression of genes involved in immune and inflammatory responses. In unstimulated cells, NF-kappaB complexes are sequestered in the cytoplasm through interactions with IkappaBalpha and other IkappaB proteins. Extracellular stimuli that activate NF-kappaB, such as tumor necrosis factor alpha (TNFalpha), cause rapid phosphorylation of IkappaBalpha at serines 32 and 36. The inducible phosphorylation of IkappaBalpha is followed by its ubiquitination and degradation, allowing NF-kappaB complexes to translocate into the nucleus and to activate gene expression. Previously, it has been shown that TNFalpha as well as other stimuli also lead to the phosphorylation of the RelA/p65 subunit of NF-kappaB. In this report, we demonstrate that the TNFalpha-induced phosphorylation of the RelA/p65 subunit occurs on serine 529, which is in the C-terminal (TA1) transactivation domain. Accordingly, the TNFalpha-induced phosphorylation of Rel/p65 increases NF-kappaB transcriptional activity but does not affect nuclear translocation or DNA binding affinity. PMID: 9792644 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 242: Biochemistry. 1998 Sep 22;37(38):13165-73. Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon-gamma, and GM-CSF and the necessity to overcome the action of endogenous proteases. McDonald PP, Bovolenta C, Cassatella MA. Department of General Pathology, University of Verona, Italy. Human neutrophils can be induced to actively transcribe a number of early-response genes, in particular those encoding cytokines, chemokines, and the high-affinity surface receptor for IgG, FcgammaRI. Although little is known to date about the regulation of gene transcription in neutrophils, several indications point to a role for distinct transcription factors, such as members of the NF-kappaB and STAT families. In this study, we investigated whether these transcription factors become activated under stimulatory conditions which are known to induce gene transcription in neutrophils. Unexpectedly, we found that conventional procedures employed to prepare cellular extracts cause the release of proteolytic activities that are normally stored in intracellular granules, resulting in the degradation of various NF-kappaB/Rel and STAT proteins. To circumvent this problem, we developed an alternative procedure which allowed us to show that in neutrophils, LPS and TNFalpha induce a NF-kappaB DNA-binding activity which essentially consists of p50/RelA dimers, and that IFNgamma promotes the binding of STAT1 homodimers to the IFNgamma response region of the FcgammaRI promoter. Moreover, we report that neutrophil stimulation with GM-CSF results in the formation of a STAT5-containing DNA-binding activity. Collectively, the current findings open new perspectives about mechanisms that are likely to regulate gene transcription in neutrophils. In addition, the procedure described herein could prove useful in other cell types that express high levels of endogenous proteases. PMID: 9748323 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 243: Mol Cell Biol. 1998 Sep;18(9):5523-32. The ability of CD40L, but not lipopolysaccharide, to initiate immunoglobulin switching to immunoglobulin G1 is explained by differential induction of NF-kappaB/Rel proteins. Lin SC, Wortis HH, Stavnezer J. Department of Molecular Genetics and Microbiology and Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122, USA. Antibodies of the immunoglobulin G1 class are induced in mice by T-cell-dependent antigens but not by lipopolysaccharide (LPS). CD40 engagement contributes to this preferential isotype production by activating NF-kappaB/Rel to induce germ line gamma1 transcripts, which are essential for class switch recombination. Although LPS also activates NF-kappaB, it poorly induces germ line gamma1 transcripts. Western blot analyses show that CD40 ligand (CD40L) induces all NF-kappaB/Rel proteins, whereas LPS activates predominantly p50 and c-Rel. Electrophoretic mobility shift assays show that in CD40L-treated cells, p50-RelA and p50-RelB dimers are the major NF-kappaB complexes binding to the germ line gamma1 promoter, whereas in LPS-treated cells, p50-c-Rel and p50-p50 dimers are the major binding complexes. Transfection of expression plasmids for NF-kappaB/Rel fusion proteins (forced dimers) indicates that p50-RelA and p50-RelB dimers activate the germ line gamma1 promoter and that p50-c-Rel and p50-p50 dimers inhibit this activation by competitively binding to the promoter without activating the promoter. Therefore, germ line gamma1 transcription depends on the composition of NF-kappaB/Rel proteins. PMID: 9710636 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 244: J Bacteriol. 1998 Aug;180(16):4123-32. The relA/spoT-homologous gene in Streptomyces coelicolor encodes both ribosome-dependent (p)ppGpp-synthesizing and -degrading activities. Martinez-Costa OH, Fernandez-Moreno MA, Malpartida F. Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autonoma de Madrid, Cantoblanco, 28049-Madrid, Spain. Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis. PMID: 9696759 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 245: Microbiology. 1998 Jul;144 ( Pt 7):1853-62. The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism. Wehmeier L, Schafer A, Burkovski A, Kramer R, Mechold U, Malke H, Puhler A, Kalinowski J. Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, Germany. To investigate the metabolism of (p)ppGpp in amino-acid-producing coryneform bacteria, a PCR-based strategy using degenerate consensus oligonucleotides was applied to isolate the rel gene of Corynebacterium glutamicum ATCC 13032. The gene consists of 2283 nucleotides and encodes a protein of 760 amino acids with a molecular mass of 84.4 kDa. The amino acid sequence revealed extensive similarities to the related proteins RelA and SpoT of Escherichia coli, which are known to be involved in (p)ppGpp biosynthesis and degradation. The C. glutamicum rel gene is located downstream of the apt gene encoding an adenine phosphoribosyltransferase, and an ORF with similarities to dciAE, which represents part of a dipeptide transport system in E. coli. A C. glutamicum mutant strain carrying a defined deletion in the rel gene was constructed. This mutant failed to accumulate (p)ppGpp in response to amino acid starvation. When overexpressed in E. coli, the C. glutamicum rel gene was able to reverse growth defects caused by an overexpressed relA gene. It is proposed that the C. glutamicum rel gene encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp-degrading activities. PMID: 9695918 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 246: Blood. 1998 Aug 15;92(4):1334-41. Impaired activation of NFkappaB in T cells from a subset of renal cell carcinoma patients is mediated by inhibition of phosphorylation and degradation of the inhibitor, IkappaBalpha. Ling W, Rayman P, Uzzo R, Clark P, Kim HJ, Tubbs R, Novick A, Bukowski R, Hamilton T, Finke J. Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. Activation of the transcription factor NFkappaB in peripheral blood T cells from patients with renal cell carcinoma (RCC) is compromised. This impaired signaling function results from a failure of RelA and c-Rel to translocate to the nucleus though normal levels of Rel proteins are present in the cytoplasm. We demonstrate here in a subset of RCC patients that the defect in NFkappaB activation is attributable to the absence of phosphorylation and degradation of the inhibitor IkappaBalpha. In patient T cells there was no stimulus dependent decrease in the cytoplasmic level of IkappaBalpha. Coimmunoprecipitation studies showed that RelA was in complex with IkappaBalpha and was not released after stimulation. Moreover, the phosphorylated form of IkappaBalpha detected in normal T cells after activation is absent in patient T cells. Additional experiments showed that soluble products from RCCs (RCC-S) can reproduce the same phenotype in T cells from healthy individuals. Supernatant fluid from cultured explants of RCC, but not normal kidney, inhibited the stimulus dependent nuclear translocation of NFkappaB without altering the cytoplasmic levels of RelA, c-Rel, and NFkappaB1. Phosphorylation and degradation of IkappaBalpha was also blocked by RCC-S. The mechanistic similarities between patient-derived T cells and normal T cells cultured with RCC-S suggest that the tumor-derived products may be the primary mediators of impaired T-cell function in this tumor system. Copyright 1998 by The American Society of Hematology. PMID: 9694722 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 247: Blood. 1998 Aug 1;92(3):990-5. Erratum in: Blood 1999 Mar 15;93(6):2141. Triggering of CD40 antigen inhibits fludarabine-induced apoptosis in B chronic lymphocytic leukemia cells. Romano MF, Lamberti A, Tassone P, Alfinito F, Costantini S, Chiurazzi F, Defrance T, Bonelli P, Tuccillo F, Turco MC, Venuta S. Dipartimento di Biochimica e Biotecnologie Mediche, Universita Federico II, Napoli, Italia. We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean +/- SE: 28.5 +/- 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% +/- 7.8% on average (P = .0077). Because the CD40 antigen activates NF-kappaB/Rel transcription factors in B cells, and NF-kappaB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-kappaB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-kappaB/Rel activity; p50, RelA, and c-Rel components of the NF-kappaB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-kappaB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-kappaB/Rel levels. To determine the involvement of NF-kappaB/Rel activity in the G28-5-mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-kappaB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-kappaB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-kappaB/Rel inhibitors, could improve the therapeutic effect of fludarabine. Copyright 1998 by The American Society of Hematology. PMID: 9680368 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 248: Mol Cell Biol. 1998 Jun;18(6):3234-44. Involvement of TFIID and USA components in transcriptional activation of the human immunodeficiency virus promoter by NF-kappaB and Sp1. Guermah M, Malik S, Roeder RG. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA. The purified Rel/NF-kappaB (p50/p65) complex and Sp1 markedly activate transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a highly purified HeLa reconstituted transcription system. Transcriptional activation by NF-kappaB and Sp1 requires both TFIID and the USA fraction. The USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator activity, both by repressing the basal level of transcription and by potentiating activator function to yield large increases in the levels of transcription induction. Under limiting concentrations, PC2 and PC4 also show synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65 subunit of NF-kappaB is a potent activator when assayed as a Gal fusion in the reconstituted transcription system and interacts both with TATA-binding protein (TBP) and with several human TBP-associated factors (TAFs) that include TAFII250. The p65 activation domain mediates transcription activation in the presence of partially reconstituted TFIID species that include a minimal complex containing only TBP and TAFII250. These studies also show that, like USA components, TAFs can serve both to repress TBP-mediated transcription and, following activator interactions, to reverse the repression and effect a net increase in activity. Taken together, these data underscore the importance of both TAFs and specific USA-derived coactivators for optimal activation of the HIV-1 promoter, as well as certain parallels in their overall mechanisms of action. PMID: 9584164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 249: J Immunol. 1998 Jun 1;160(11):5374-81. Overexpression of p65 and c-Jun substitutes for B7-1 costimulation by targeting the CD28RE within the IL-2 promoter. Parra E, McGuire K, Hedlund G, Dohlsten M. Department of Cell and Molecular Biology, University of Lund, Sweden. The role of Rel and activation protein-1 (AP-1) in IL-2 promoter activity in B7-1- and leukocyte function-associated Ag-3 (LFA. 3)-costimulated T cells has been evaluated. We demonstrate that overexpression of c-Jun but not c-Fos increases IL-2 promoter activity in both B7-1- and LFA-3-costimulated Jurkat T cells. Cotransfection of both c-Jun and c-Fos substitutes for B7-1 costimulation in driving an activation protein-1 response element but not for the IL-2 promoter. Overexpression of Rel proteins demonstrated that p65-expressing Jurkat cells transcribed equally well a nuclear factor kappabeta reporter construct when costimulated with B7-1 or LFA-3, but transcription of IL-2 promoter or CD28 response element (CD28RE)-driven reporters was superior in B7-1-costimulated cells. Combined expression of c-Jun and p65 induced vigorous transcription of IL-2 promoter- and CD28RE-driven reporter constructs in both LFA-3- and B7-1-costimulated Jurkat cells. Mutating the CD28RE but not the upstream nuclear factor kappabeta-binding site in the IL-2 promoter reduced B7-1-driven transcription >90%. The results implicates a major role of the CD28RE in the integration of p65/c-Jun-mediated transcription within the IL-2 promoter. We suggest that the transition from an autocrine LFA-3-driven immune response to a B7--induced paracrine immune response involves the activation of c-Jun and p65, which target the CD28RE region of the IL-2 promoter. PMID: 9605137 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 250: Blood. 1998 Jun 1;91(11):4136-44. NF-kappaB transcription factors are involved in normal erythropoiesis. Zhang MY, Sun SC, Bell L, Miller BA. Department of Pediatrics, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033-0850, USA. NF-kappaB/Rel designates a widely distributed family of transcription factors involved in immune and acute phase responses. Here, the expression and function of NF-kappaB factors in erythroid proliferation and differentiation were explored. In an erythroleukemia cell line, TF-1, high levels of p105/p50, p100/p52, p65, and IkappaBalpha were detected 24 hours after growth factor deprivation. In response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, significant induction of p52 expression was observed. GM-CSF also induced nuclear translocation of both p52 and p65. No induction of NF-kappaB factors was observed with erythropoietin stimulation of TF-1 cells. Overexpression of p52 and p65 in TF-1 cells by transient transfection resulted in significant induction of a kappaB-TATA-luciferase reporter plasmid, showing that these factors are functional in vivo in erythroid cells. To determine whether NF-kappaB factors may play a role in normal erythropoiesis, levels of these factors were determined in burst-forming unit-erythroid (BFU-E)-derived cells at different stages of differentiation. The NF-kappaB factors p105/p50, p100/p52, and p65 were highly expressed in early BFU-E-derived precursors, which are rapidly proliferating, and declined during maturation. Furthermore, nuclear levels of NF-kappaB factors p50, p52, and p65 were higher in less mature precursors (day 10 BFU-E-derived cells) compared with more differentiated (day 14) erythroblasts. In nuclear extracts from day 10 BFU-E-derived cells, p50, p52, and p65 were able to form complexes, which bound to kappaB sites in the promoters of both the c-myb and c-myc genes, suggesting that c-myb and c-myc may be among the kappaB-containing genes regulated by NF-kappaB factors in normal erythroid cells. Taken together, these data show that NF-kappaB factors are modulated by GM-CSF and suggest they function to regulate specific kappaB containing genes involved in erythropoiesis. PMID: 9596659 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 251: J Neuropathol Exp Neurol. 1998 Feb;57(2):168-78. Transcription factor NF-kappaB and inhibitor I kappaBalpha are localized in macrophages in active multiple sclerosis lesions. Gveric D, Kaltschmidt C, Cuzner ML, Newcombe J. Multiple Sclerosis Laboratory, Institute of Neurology, London, England. NF-kappaB is a transcription factor family which on translocation to the nucleus regulates gene expression during cell activation. As such, NF-kappaB may play a role in the microglial response to myelin damage in multiple sclerosis (MS) lesions. Here the cellular localization of NF-kappaB and expression of the inhibitory I kappaBalpha were examined by immunocytochemistry on central nervous system (CNS) tissue from MS and control cases. In normal control white matter, the active form of the NF-kappaB subunit RelA (p65) was localized in microglial nuclei, while the c-Rel and p50 subunits and the inhibitory I kappaBalpha were restricted to the cytoplasm. In contrast, in actively demyelinating plaques, the RelA, c-Rel, and p50 subunits of NF-kappaB and I kappaBalpha were all present in macrophage nuclei in both parenchymal and perivascular areas. RelA was also found in the nuclei of a subset of hypertrophic astrocytes. Only c-Rel had a nuclear localization in lymphocytes in perivascular inflammatory cuffs. Our results suggest that constitutive activation of the RelA subunit in the nuclei of resting microglia may facilitate a rapid response to pathological stimuli in the CNS. Activation of the inducible NF-kappaB pool in macrophages in MS lesions could amplify the inflammatory reaction through upregulation of NF-kappaB-controlled adhesion molecules and cytokines. PMID: 9600209 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 252: Arch Biochem Biophys. 1998 Apr 1;352(1):59-70. Protein tyrosine kinase inhibitors block tumor necrosis factor-induced activation of nuclear factor-kappaB, degradation of IkappaBalpha, nuclear translocation of p65, and subsequent gene expression. Natarajan K, Manna SK, Chaturvedi MM, Aggarwal BB. Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. Several inflammatory effects of tumor necrosis factor (TNF) are known to be mediated through activation of a nuclear transcription factor NF-kappaB, but how TNF activates NF-kappaB is incompletely understood. In the present report, we examined the role of protein tyrosine kinases (PTK) in TNF-mediated NF-kappaB activation by using genistein and erbstatin, two potent inhibitors of PTK. The treatment of human myeloid U-937 cells with either inhibitor completely suppressed the TNF-induced NF-kappaB activation in a dose- and time-dependent manner. Suppression correlated with PTK activity, since among the structural analogues of genistein, only an active inhibitor of PTK, quercetin blocked TNF-induced NF-kappaB activation and not daidzein, an inactive inhibitor. Inhibition of NF-kappaB activation was not limited to myeloid cells, as it was observed with T cells and epithelial cells. Both the PTK inhibitors blocked the degradation of IkappaBalpha, the inhibitory subunit of NF-kappaB, and the consequent translocation of the p65 subunit without any significant effect on p50 or on c-Rel. The PTK inhibitors did not interfere with NF-kappaB binding to DNA. The NF-kappaB-dependent CAT reporter gene expression in transient transfection assays was also suppressed by the PTK inhibitors. Both PTK inhibitors abolished TNF-induced activation of N-terminal c-Jun kinase and mitogen-activated protein kinase kinase. Overall, our results suggest that a genistein- and erbstatin-sensitive PTK is involved in the pathway leading to NF-kappaB activation and gene expression by TNF and thus could be used as a target for development of antiinflammatory drugs. Copyright 1998 Academic Press. PMID: 9521814 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 253: Oncogene. 1998 Apr 23;16(16):2033-9. Activation of the wt1 Wilms' tumor suppressor gene by NF-kappaB. Dehbi M, Hiscott J, Pelletier J. Department of Biochemistry, McGill University, Montreal, Quebec, Canada. The Wilm's tumor suppressor gene, wt1, is expressed in a very defined spatial-temporal fashion and plays a key role in development of the urogenital system. Transacting factors governing wt1 expression are poorly defined. The presence of putative kappa-B binding sites within the wt1 gene prompted us to investigate whether members of the NF-kappaB/Rel family of transcription factors are involved in regulating wt1 expression. In transient transfection assays, ectopic expression of p50 and p65 subunits of NF-kappaB stimulated wt1 promoter activity 10-30-fold. Deletion mutagenesis revealed that NF-kappa-B responsiveness is mediated by a short DNA fragment located within promoter proximal sequences of the major transcription start site. Two kappaB-binding sites are present in this region and form specific complexes with purified NF-kappaB proteins, as revealed by electrophoretic mobility gel shift assays. Ectopic expression of p50 and p65 resulted in increased transcription of the endogenous wt1 gene, as revealed by nuclear run-on experiments. Taken together, these results indicate that members of the NF-kappaB/Rel family are important for activating expression of wt1 and reside upstream of the regulatory cascade leading to wt1 activation. PMID: 9572484 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 254: Int J Biochem Cell Biol. 1997 Dec;29(12):1525-39. Cross-talk between transcription factors NF-kappa B and C/EBP in the transcriptional regulation of genes. Xia C, Cheshire JK, Patel H, Woo P. Department of Molecular Pathology, University College London, U.K. The study of the acute phase response has attracted substantial interest, not only for its medical implication, but also its provision as an excellent system with which to elucidate the molecular mechanisms involved in the modulation of gene expression. Our previous data suggest that the synergistic induction of the major acute phase reactant serum amyloid A2 (SAA2) expression by interleukin-1 (IL-1) and interleukin-6 (IL-6) is mediated by two families of transcription factors, namely NF-kappa B and C/EBP. To understand the molecular mechanisms of this synergy, we have undertaken a molecular dissection of the factors involved in the formation of the regulatory complex. Electrophoretic mobility shift analysis indicates that NF-kappa B p65 (RelA) and p50, but not p52 or c-Rel, bind specifically to the NF-kappa B site of the SAA2 promoter in response to IL-1 stimulation. In addition, C/EBP beta and C/EBP delta, but not C/EBP alpha, bind specifically to the C/EBP site of SAA2 in response to IL-6 stimulation. Transient co-transfection analysis indicates that co-operative association of NF-kappa B p65 with C/EBP beta and, in particular, with C/EBP delta, results in synergistic transcriptional activation of the SAA2 promoter. When incubated together, NF-kappa B p65 and C/EBP beta form a ternary complex by direct protein/protein interaction. Mutational analysis demonstrates that the C-terminus region of the Rel homology domain (RHD) and the C-terminus of the activation domain of p65 are important for its interaction with C/EBP beta. These results suggest the NF-kappa B and C/EBP may form a new complex of transcription factors that mediates the synergistic induction of SAA2 by IL-1 and IL-6. PMID: 9570146 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 255: Mol Cell Biol. 1998 May;18(5):2640-9. The N-terminal domain of IkappaB alpha masks the nuclear localization signal(s) of p50 and c-Rel homodimers. Latimer M, Ernst MK, Dunn LL, Drutskaya M, Rice NR. Molecular Basis of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21701, USA. Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB) which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IkappaB alpha and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha, (ii) the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IkappaB alpha, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs. PMID: 9566883 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 256: Mol Cell Biol. 1998 May;18(5):2524-34. Nuclear localization of IkappaB alpha is mediated by the second ankyrin repeat: the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences. Sachdev S, Hoffmann A, Hannink M. Biochemistry Department, University of Missouri-Columbia, 65212, USA. The ability of the IkappaB alpha protein to sequester dimeric NF-kappaB/Rel proteins in the cytoplasm provides an effective mechanism for regulating the potent transcriptional activation properties of NF-kappaB/Rel family members. IkappaB alpha can also act in the nucleus as a postinduction repressor of NF-kappaB/Rel proteins. The mechanism by which IkappaB alpha enters the nucleus is not known, as IkappaB alpha lacks a discernible classical nuclear localization sequence (NLS). We now report that nuclear localization of IkappaB alpha is mediated by a novel nuclear import sequence within the second ankyrin repeat. Deletion of the second ankyrin repeat or alanine substitution of hydrophobic residues within the second ankyrin repeat disrupts nuclear localization of IkappaB alpha. Furthermore, a region encompassing the second ankyrin repeat of IkappaB alpha is able to function as a discrete nuclear import sequence. The presence of a discrete nuclear import sequence in IkappaB alpha suggests that cytoplasmic sequestration of the NF-kappaB/Rel-IkappaB alpha complex is a consequence of the mutual masking of the NLS within NF-kappaB/Rel proteins and the import sequence within IkappaB alpha. Nuclear import may be a conserved property of ankyrin repeat domains (ARDs), as the ARDs from two other ARD-containing proteins, 53BP2 and GABPbeta, are also able to function as nuclear import sequences. We propose that the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences. PMID: 9566872 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 257: Cancer Lett. 1998 Mar 13;125(1-2):157-64. Enhancement by antisense oligonucleotides to NF-kappaB of the differentiation of HL-60 promyelocytic leukemia cells induced by vitamin D3. Sokoloski JA, Narayanan R, Sartorelli AC. Department of Pharmacology and Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA. We have demonstrated previously that a phosphorothioate antisense oligonucleotide to the p65 subunit of the inducible transcription factor NF-kappaB produced rapid changes in the expression of leukocyte integrin CD11b (Mo 1) and in the adhesion of dimethylsulfoxide (DMSO)-differentiated HL-60 cells stimulated by 12-O-tetradecanoylphorbol 13-acetate. We have also shown that a variety of agents which inhibit NF-kappaB, including vitamin E and related antioxidants, curcumin and several non-steroidal anti-inflammatory agents, significantly enhanced the differentiation of HL-60 leukemia cells when combined with low levels of 1,25-dihydroxyvitamin D3 (vitamin D3). To provide further evidence that interference with the activation of NF-kappaB affects the maturation of HL-60 leukemia cells by creating an environment conducive to terminal differentiation, we measured the effects of phosphorothioate antisense oligonucleotides to the various subunits of NF-kappaB on the differentiation of HL-60 cells produced by low levels of vitamin D3. When used alone these oligonucleotides had no significant effect on the differentiation of HL-60 cells. However, the antisense oligomer to the Rel A subunit of NF-kappaB markedly increased the extent of differentiation produced by low levels of vitamin D3. An enhancement of the differentiation of HL-60 cells induced by vitamin D3 was also obtained by several transcription factor decoys designed to mimic the consensus sequences of genes activated by Rel A. The findings provide additional support for the concept that inhibition of the activation of NF-kappaB may be involved in regulating the entry of promyelocytic leukemia cells into a differentiation pathway. PMID: 9566710 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 258: J Immunol. 1997 Dec 1;159(11):5620-8. Thrombin activates nuclear factor-kappaB and potentiates endothelial cell activation by TNF. Anrather D, Millan MT, Palmetshofer A, Robson SC, Geczy C, Ritchie AJ, Bach FH, Ewenstein BM. Sandoz Center for Immunobiology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA. Thrombin is the central bioregulatory enzyme in hemostasis and is generated in vascular beds in which inflammatory responses are ongoing. In this study, we examined the effect of thrombin, both alone and in combination with TNF, on gene expression in porcine aortic endothelial cells (EC). Thrombin (1-10 U/ml) induced increased mRNA levels of E-selectin, monocyte chemoattractant protein-1, IL-8, plasminogen activator inhibitor-1, and IkappaB-alpha. These effects were mimicked by a thrombin receptor-activating peptide; preincubation of thrombin with hirudin blocked the induction of mRNA, suggesting that the increased gene expression was due to thrombin-specific activity. Because these genes are known to contain nuclear-factor-kappaB (NF-kappaB)-binding elements in their promoter region, we next examined the ability of thrombin to activate this transcription factor. As detected by electrophoretic mobility shift assay, thrombin (10 U/ml) or thrombin receptor-activating peptide (100 microM) stimulated increased NF-kappaB-binding activity. Supershift analysis revealed that these complexes were comprised principally of the RelA (p65) and NF-kappaB1 (p50) Rel family members. Thrombin alone did not substantively increase protein levels of E-selectin despite the increase in E-selectin mRNA levels. However, thrombin (3-10 U/ml) stimulated a 10-fold enhancement in the ability of TNF (0.3-1.0 ng/ml) to induce E-selectin surface expression. Similar potentiation of TNF-induced NF-kappaB activity and E-selectin transcription by thrombin was observed in experiments utilizing luciferase reporter constructs expressed in bovine aortic EC. The ability of thrombin to potentiate TNF-induced EC activation thus provides an important mechanism by which products of the coagulation cascade may enhance cytokine-mediated inflammatory responses. PMID: 9548505 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 259: Cell. 1998 Mar 20;92(6):819-28. Cotranslational biogenesis of NF-kappaB p50 by the 26S proteasome. Lin L, DeMartino GN, Greene WC. Gladstone Institute of Virology and Immunology, Department of Microbiology and Immunology, University of California, San Francisco 94141, USA. The NFkappaB1 gene encodes two functionally distinct proteins termed p50 and p105. p50 corresponds to the N terminus of p105 and with p65 (RelA) forms the prototypical NF-kappaB transcription factor complex. In contrast, p105 functions as a Rel-specific inhibitor (IKB) and has been proposed to be the precursor of p50. Our studies now demonstrate that p50 is generated by a unique cotranslational processing event involving the 26S proteasome, whereas cotranslational folding of sequences near the C terminus of p50 abrogates proteasome processing and leads to p105 production. These results indicate that p105 is not the precursor of p50 and reveal a novel mechanism of gene regulation that ensures the balanced production and independent function of the p50 and p105 proteins. PMID: 9529257 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 260: Mol Cell Biol. 1998 Apr;18(4):2077-88. NF-kappaB2 is a putative target gene of activated Notch-1 via RBP-Jkappa. Oswald F, Liptay S, Adler G, Schmid RM. Department of Internal Medicine, University of Ulm, Germany. NF-kappaB2 (p100/p52), a member of the NF-kappaB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-kappaB2 gene is regulated in a positive and a negative manner. Two kappaB elements within the NF-kappaB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-kappaB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-kappaB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-kappaB-DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-kappaB binding activity is recognized by an antibody directed against RBP-Jkappa. By performing transient-transfection assays, we show that human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappaB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-kappaB2 promoter activity. In summary, our data clearly demonstrate that Rep-kappaB is closely related or identical to RBP-Jkappa. RBP-Jkappa is a strong transcriptional repressor of NF-kappaB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-kappaB2 is a novel putative Notch target gene. PMID: 9528780 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 261: J Immunol. 1998 Mar 15;160(6):2872-80. Superinduction of IL-8 in T cells by HIV-1 Tat protein is mediated through NF-kappaB factors. Ott M, Lovett JL, Mueller L, Verdin E. The Picower Institute for Medical Research, Manhasset, NY 11030, USA. Elevated levels of circulating IL-8, a potent chemotactic factor for granulocytes and T lymphocytes, are found in HIV-infected individuals. The HIV-1 transactivator protein Tat increased IL-8 secretion in T cell lines following CD3- and CD28-mediated costimulation. Full-length Tat (Tat101) enhanced IL-8 transcription through up-regulated transcription factor binding to the CD28-responsive element (CD28RE) in the IL-8 promoter. Expression of the Tat splice variant Tat72 (72 amino acids) also enhanced IL-8 production following T cell stimulation via a different, most likely post-transcriptional, mechanism. The CD28RE in the IL-8 promoter was characterized as a low-affinity NF-kappaB binding site recognized by the transcription factors p50 (NF-kappaB1), p65 (RelA) and c-rel. Transcription factor binding to "classical" NF-kappaB sites in the HIV-1, the human IL-2, and lymphotoxin promoters, recognized by p50 and p65 following CD3+28-mediated costimulation, was unaffected by Tat101 as was binding to the AP-1 motif in the IL-8 promoter. These experiments identify the CD28RE in the IL-8 promoter as a c-rel recognition site and a Tat101-responsive element. The effect of Tat101 on CD28REs in the IL-8 promoter and the subsequent up-regulation of IL-8 secretion is likely to contribute to the immune dysregulation observed during HIV-1 infection. PMID: 9510190 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 262: Cancer Res. 1998 Mar 1;58(5):882-6. CD95 (Fas)-induced caspase-mediated proteolysis of NF-kappaB. Ravi R, Bedi A, Fuchs EJ, Bedi A. Johns Hopkins Oncology Center, Division of Experimental Therapeutics and Pharmacology, Baltimore, Maryland 21287, USA. Activation of the nuclear factor (NF)-kappaB transcription factor is instrumental for the immune response and the survival of peripheral activated T cells. We demonstrate that ligation of CD95 (Fas/APO1), a potent apoptotic stimulus in lymphocytes, results in repression of NF-kappaB activity in Jurkat T cells by inducing the proteolytic cleavage of NF-kappaB p65 (Rel A) and p50. Inhibition of caspase-3-related proteases by a specific acetylated aldehyde (Ac-DEVD-CHO) prevented CD95-induced cleavage of p65 (RelA) or p50 and restored the inducibility of NF-kappaB in cells treated with an antibody against CD95. The addition of recombinant caspase-3 also resulted in proteolytic cleavage of RelA p65 and p50 in vitro. TNF-alpha treatment, unlike CD95 ligation, did not result in the death of Jurkat cells but did so in the presence of I kappaB alphaM, a transdominant inhibitor of NF-kappaB. These results suggest that intact, functional NF-kappaB maintains the survival of activated T cells, and that CD95-induced cleavage of NF-kappaB subunits sensitizes T cells to apoptosis and, hence, facilitates the decay of an immune response. PMID: 9500443 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 263: J Immunol. 1998 Mar 1;160(5):2308-17. Induction of nuclear factor-kappa B during primary B cell differentiation. Kistler B, Rolink A, Marienfeld R, Neumann M, Wirth T. MSZ, Institut fur Medizinische Strahlenkunde und Zellforschung, Universitat Wurzburg, Germany. We have investigated activation of nuclear factor-kappa B (NF-kappa B) in the process of primary B cell differentiation in vitro. In this system, NF-kappa B is strongly induced when B cells develop from the pre-B cell to the immature B cell stage. Unlike the typical NF-kappa B activation in response to exogenous stimuli, induction proceeds with a slow time course. NF-kappa B induction is only observed in B cells that undergo differentiation, not in Rag2-deficient cells. Nuclear DNA binding complexes predominantly comprise p50/RelA heterodimers and, to a lesser extent, c-Rel-containing dimers. The increase in NF-kappa B binding activity is accompanied by a slow and steady decrease in I kappa B beta protein levels. Interestingly, absolute RelA protein levels remain unaffected, whereas RelB and c-Rel synthesis is induced. The reason for preferential nuclear translocation of RelA complexes appears to be selective inhibition by the I kappa B beta protein. I kappa B beta can efficiently inhibit p50/RelA complexes, but has a much reduced ability to interfere with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly, p50/RelB complexes are not at all targeted by I kappa B beta, and coimmunoprecipitation experiments show no evidence for an association of I kappa B beta and RelB in vivo. Consistent with these observations, I kappa B beta cotransfection can inhibit p50/RelA-mediated trans-activation, but barely affects p50/RelB mediated trans-activation. PMID: 9498771 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 264: Int J Dev Biol. 1998 Jan;42(1):67-77. Involvement of NF-kappaB associated proteins in FGF-mediated mesoderm induction. Beck CW, Sutherland DJ, Woodland HR. Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. bsscb@bath.ac.uk In this report, we have used mRNA injection to study the action of mutants of XrelA, a Xenopus homolog of the RelA (p65) component of NF-kappaB, on the induction of mesoderm in Xenopus embryos. A region of the rel homology domain of XrelA was deleted to create XrelA deltaSP, which retains the dimerization and activation domains, but no longer binds to DNA. We also made an analogous derivative of mammalian NF-kappaB1 (p50). We show that both constructs have dominant inhibitory activity. When message encoding either is injected into eggs or oocytes, DNA binding of rel family members is suppressed, as is transactivation of a kappaB-dependent promoter in embryos. Expression of XrelA deltaSP in animal caps blocks the induction of mesoderm by bFGF. In addition, this mutant prevents elongation movements generated by activin, but has little effect on posterior dorsal cytodifferentiation, which in marked contrast is blocked by inhibition of the FGF signal transduction pathway between the receptor and MAP kinase. The specificity of the XrelA deltaSP effect on FGF signaling is shown by rescue of mesodermal marker expression when XrelA deltaSP is co-expressed with a specific rel inhibitor. The target of these dominant negative constructs seems to be neither XrelA itself, nor p50, but rather some other molecule with which XrelA, rather than NF-kappaB1, heterodimerizes. We show that XrelA deltaSP blocks FGF induction of mesoderm downstream of MAP kinase and Xbra expression. Thus it prevents the maintenance of Xbra expression by inhibiting its autoregulation by embryonic FGF (eFGF). We suggest that XrelA deltaSP differs from other reported inhibitors of FGF signaling because it inhibits only gastrula stage FGF signaling and not the maternally programmed signaling at the blastula stage. Our results therefore suggest that zygotic FGF action is required for cell movements rather than dorsal differentiation. PMID: 9496788 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 265: Immunogenetics. 1998;47(3):196-205. Expression of CD14 corrects the slow response to lipopolysaccharide in the 1B8 mutant of the B cell lymphoma 70Z/3. Brophy VH, Sibley CH. Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195, USA. B cells and macrophages both activate NF-kappaB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14+ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IkappaB-alpha and -beta molecules and translocation of the NF-kappaB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14+ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IkappaB-beta was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself. PMID: 9435337 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 266: Structure. 1997 Nov 15;5(11):1427-36. The role of DNA in the mechanism of NFkappaB dimer formation: crystal structures of the dimerization domains of the p50 and p65 subunits. Huang DB, Huxford T, Chen YQ, Ghosh G. Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA. BACKGROUND: Members of the rel/NFkappaB family of transcription factors play a vital role in the regulation of rapid cellular responses, such as those required to fight infection or react to cellular stress. Members of this family of proteins form homo- and heterodimers with differing affinities for dimerization. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Crystal structures of the rel/NFkappaB family members p50 and p65 in their DNA-bound homodimeric form have been solved. These structures showed that the residues from the dimerization domains of both p50 and p65 participate in DNA binding and that the DNA-protein and protein dimerization surfaces form one continuous overlapping interface. We desired to investigate the contribution of DNA to NFkappaB dimerization and to identify the mechanism for the selective association of rel/NFkappaB family peptides into transcriptionally active dimers. RESULTS: We report here the crystal structures of the dimerization domains of murine p50 and p65 at 2.2 A and 2.0 A resolution, respectively. A comparison of these two structures suggests that conservative amino acid changes at three positions are responsible for the differences in their dimer interfaces. The presence of the target DNA does not change the dimer interface of either protein in any significant manner. CONCLUSIONS: These two structures suggest that the rel/NFkappaB family of transcription factors use only a few conservative changes in their amino acid sequences to form a host of dimers with varying affinities for dimerization. Amino acids at positions corresponding to 254, 267, and 307 of murine p50, function as primary determinants for the observed differences in dimerization affinity. The DNA-contacting charged amino acid sidechains from the dimerization domains are held in a similar conformation in both the DNA-bound and free states, therefore, no major structural rearrangement is required to bring these residues into contact with the DNA. PMID: 9384558 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 267: Mol Cell Biol. 1998 Mar;18(3):1266-74. Functional interference of Sp1 and NF-kappaB through the same DNA binding site. Hirano F, Tanaka H, Hirano Y, Hiramoto M, Handa H, Makino I, Scheidereit C. Max Delbruck Center for Molecular Medicine MDC, Berlin, Germany. Gene activation by NF-kappaB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-kappaB through nonoverlapping binding sites. Here we report that Sp1 acts directly through a subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these NF-kappaB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is in the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does not bind to a GC box Sp1 site. Sp1 can activate transcription through immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus provides a means to keep an elevated basal expression of NF-kappaB-dependent genes in the absence of activated nuclear NF-kappaB/Rel. PMID: 9488441 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 268: J Exp Med. 1998 Jan 19;187(2):143-6. Erratum in: J Exp Med 1998 Feb 16;187(4):661. Regulation of immune responses by NF-kappa B/Rel transcription factor. Sha WC. Molecular and Cell Biology Department, University of California, Berkeley 94720-3200, USA. bsha@uclink4.berkeley.edu Publication Types: Review Review, Tutorial PMID: 9432972 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 269: J Biol Chem. 1998 Jan 2;273(1):592-9. Discrimination between RelA and RelB transcriptional regulation by a dominant negative mutant of IkappaBalpha. Ferreira V, Tarantino N, Korner M. Laboratoire d'Immunologie Cellulaire, CNRS URA 625, Bat. CERVI, Hopital de la Pitie Salpetriere, 83, Bd. de l'Hopital, 75013 Paris, France. RelA and RelB belong to the nuclear factor-kappaB (NF-kappaB-Rel) transcription factor family. Both proteins are structurally and functionally related, but their intracellular and tissue distributions are different. In resting cells, RelB is found mostly in the nucleus, whereas RelA is sequestered in the cytosol by protein inhibitors, among which IkappaBalpha is the dominant form in lymphocytes. Upon cellular activation IkappaBalpha is proteolyzed, allowing RelA dimers to enter the nucleus and activate target genes. To study the selectivity of gene regulation by RelA and RelB, we generated T cell lines stably expressing a dominant negative mutant of IkappaBalpha. We show that selective inhibition of RelA-NF-kappaB decreased induction of NFKB1, interleukin-2, and interleukin-2Ralpha genes but not c-myc. Transcription driven by the IkappaBalpha promoter was blocked by the transgenic IkappaBalpha; however, wild type IkappaBalpha was expressed in the transgenic cell clones but with much slower kinetics than that in control cells. Wild type IkappaBalpha expression was concomitant with RelB up-regulation, suggesting that RelB could be involved in transcription of IkappaBalpha through binding to an alternative site. These results indicate that RelB and RelA have both distinct and overlapping effects on gene expression. PMID: 9417120 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 270: Nature. 1998 Jan 22;391(6665):410-3. Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Chen FE, Huang DB, Chen YQ, Ghosh G. Department of Biology, University of California, San Diego, La Jolla 92093-0359, USA. The NF-kappaB p50/p65 heterodimer is the classical member of the Rel family of transcription factors which regulate diverse cellular functions such as immune response, cell growth, and development. Other mammalian Rel family members, including the proteins p52, proto-oncoprotein c-Rel, and RelB, all have amino-terminal Rel-homology regions (RHRs). The RHR is responsible for the dimerization, DNA binding and cytosolic localization of these proteins by virtue of complex formation with inhibitor kappaB proteins. Signal-induced removal of kappaB inhibitors allows translocation of dimers to the cell nucleus and transcriptional regulation of kappaB DNA-containing genes. NF-kappaB specifically recognizes kappaB DNA elements with a consensus sequence of 5'-GGGRNYYYCC-3' (R is an unspecified purine; Y is an unspecified pyrimidine; and N is any nucleotide). Here we report the crystal structure at 2.9 A resolution of the p50/p65 heterodimer bound to the kappaB DNA of the intronic enhancer of the immunoglobulin light-chain gene. Our structure reveals a 5-base-pair 5' subsite for p50, and a 4-base-pair 3' subsite for p65. This structure indicates why the p50/p65 heterodimer interface is stronger than that of either homodimer. A comparison of this structure with those of other Rel dimers reveals that both subunits adopt variable conformations in a DNA-sequence-dependent manner. Our results explain the different behaviour of the p50/p65 heterodimer with heterologous promoters. PMID: 9450761 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 271: J Clin Invest. 1997 Dec 15;100(12):2952-60. Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. Sovak MA, Bellas RE, Kim DW, Zanieski GJ, Rogers AE, Traish AM, Sonenshein GE. Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA. Expression of nuclear factor-kappaB (NF-kappaB)/Rel transcription factors has recently been found to promote cell survival, inhibiting the induction of apoptosis. In most cells other than B lymphocytes, NF-kappaB/Rel is inactive, sequestered in the cytoplasm. For example, nuclear extracts from two human untransformed breast epithelial cell lines expressed only very low levels of NF-kappaB. Unexpectedly, nuclear extracts from two human breast tumor cell lines displayed significant levels of NF-kappaB/Rel. Direct inhibition of this NF-kappaB/ Rel activity in breast cancer cells induced apoptosis. High levels of NF-kappaB/Rel binding were also observed in carcinogen-induced primary rat mammary tumors, whereas only expectedly low levels were seen in normal rat mammary glands. Furthermore, multiple human breast cancer specimens contained significant levels of nuclear NF-kappaB/Rel subunits. Thus, aberrant nuclear expression of NF-kappaB/Rel is associated with breast cancer. Given the role of NF-kappaB/Rel factors in cell survival, this aberrant activity may play a role in tumor progression, and represents a possible therapeutic target in the treatment of these tumors. PMID: 9399940 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 272: Immunobiology. 1997 Dec;198(1-3):73-80. p50 (NF-kappa B1) is upregulated in LPS tolerant P388D1 murine macrophages. Ziegler-Heitbrock HW, Petersmann I, Frankenberger M. Institute of Immunology, University of Munich, Germany. ziegler@ifi.med.uni-muenchen.de Cells of the murine macrophage cell line P388D1 express cell surface CD14 and respond to LPS (lipopolysaccharide) stimulation with the production of TNF (tumor necrosis factor). When the cells are stimulated with LPS a second time then little TNF is produced, i.e. the cells are tolerant. Flow cytometry analysis demonstrates that this tolerance is not due to a downregulation of the CD14 cell surface receptor. Analysis of proteins binding to the -516 NF-kappa B motif of the murine TNF promoter reveals that constitutive p50p50 and LPS stimulation lead to mobilization of a heterodimer consisting of p65/c-rel. In tolerant cells less of the p65/c-rel heterodimer is mobilized but there is a strong upregulation of p50p50. These data show that tolerance to LPS in murine macrophages may involve a predominance of p50 homodimers. PMID: 9442379 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 273: Immunobiology. 1997 Dec;198(1-3):65-72. The transcriptional silencer protein NRF: a repressor of NF-kappa B enhancers. Nourbakhsh M, Hauser H. Department of Gene Regulation and Differentiation, GBF-Gesellschaft fur Biotechnologische Forschung mbH, Braunschweig, Germany. NF-kappa B/rel proteins are present in most cell types. In concert with other transcriptional factors they regulate a variety of genes which contribute to a wide spectrum of physiological activities like inflammation and apoptosis. An excellent example of this combinatorial regulation takes place in the IFN-beta promoter. In this promoter the fundamental regulatory elements are assembled within less than 100 base pairs including a NF-kappa B/rel enhancer and a negative regulatory element, called NRE. NRE is a member of a new class of transcriptional repressor sequences with a silencing capacity targeted to the NF-kappa B/rel enhancer. NRF is a novel transcriptional factor that binds to NRE. NRF belongs to a major class of transcriptional repressors that interact with specific promoter elements and repress transcription by separable repression domains. Such molecules have been termed active repressors, because they act by inhibitory protein-protein interaction and not simply by steric hindrance. PMID: 9442378 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 274: Immunobiology. 1997 Dec;198(1-3):50-64. NF-kappa B/Rel family members regulating the ICAM-1 promoter in monocytic THP-1 cells. Wissink S, van de Stolpe A, Caldenhoven E, Koenderman L, van der Saag PT. Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Nijmegen, The Netherlands. A kappa B-site was identified in the promoter of the intercellular adhesion molecule-1 (ICAM-1) gene, which is involved in regulation of ICAM-1 expression by tumor necrosis factor alpha (TNF-alpha) and glucocorticoids. We now report on the transcription factors which bind and transactivate this enhancer sequence. In vitro, the ICAM-1 kappa B site appeared to bind RelA and c-Rel homodimers as well as heterodimers with NF-kappa B1, but weakly NF-kappa B1 homodimers. In addition, both RelA and c-Rel, but not NF-kappa B1, were shown to transactivate an ICAM-1 kappa B-reporter construct. In monocytic THP-1 cells TNF-alpha induced two nuclear complexes which in vitro bound to the ICAM-1 kappa B site. Using antibodies in an electrophoretic mobility supershift assay, one of these complexes was shown to contain NF-kappa B1 and RelA, and to bind with higher affinity to the consensus kappa B site in the HIV long terminal repeat. The second complex contained RelA, and exhibited higher affinity towards the ICAM-1 kappa B than to the HIV kappa B site. The glucocorticoid receptor was shown to repress activity of both the RelA homodimer and the NF-kappa B1/RelA heterodimer. We argue that in vivo RelA homodimers are likely to play a dominant role in TNF-alpha-induced ICAM-1 transcription in monocytic cells. PMID: 9442377 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 275: Biol Chem. 1997 Nov;378(11):1237-45. Multiple redox regulation in NF-kappaB transcription factor activation. Piette J, Piret B, Bonizzi G, Schoonbroodt S, Merville MP, Legrand-Poels S, Bours V. Laboratory of Virology, Institute of Pathology, University of Liege, Belgium. The well-known Rel/NF-kappaB family of vertebrate transcription factors comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or hetero-dimers. All Rel proteins contain a highly conserved domain of approximately 300 amino-acids, called the Rel homology domain (RH), which contains sequences necessary for the formation of dimers, nuclear localization, DNA binding and IkappaB binding. Nuclear expression and consequent biological action of the eukaryotic NF-kappaB transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as IkappaB. The IkappaB proteins include a group of related proteins that interact with Rel dimers and regulate their activities. The interaction of a given IkappaB protein with a Rel complex can affect the Rel complex in distinct ways. In the best characterized example, IkappaB-alpha interacts with a p50/RelA (NF-kappaB) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. The NF-kappaB/IkappaB-alpha complex is located in the cytoplasm of most resting cells, but can be rapidly induced to enter the cell nucleus. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), IkappaB-alpha undergoes phosphorylation at serine residues by a ubiquitin-dependent protein kinase, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, probably while still complexed with NF-kappaB. Removal of IkappaB-alpha uncovers the nuclear localization signals on subunits of NF-kappaB, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. Like proinflammatory cytokines (e.g. IL-1, TNF), various ROS (peroxides, singlet oxygen, ...) as well as UV (C to A) light are capable of mediating NF-kappaB nuclear translocation, while the sensor molecules which are sensitive to these agents and trigger IkappaB-alpha proteolysis are still unidentified. We also show that a ROS-independent mechanism is activated by IL-1beta in epithelial cells and seems to involve the acidic sphingomyelinase/ceramide transduction pathway. Publication Types: Review Review, Tutorial PMID: 9426183 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 276: J Biol Chem. 1997 Dec 5;272(49):31092-9. Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. Cooperation of two NF-kappaB sites and NF-kappaB/Rel subunit specificity. Ueda A, Ishigatsubo Y, Okubo T, Yoshimura T. Immunopathology Section, Laboratory of Immunobiology, National Cancer Institute-Frederick, Cancer Research and Development Center, Frederick, Maryland 21702, USA. Human monocyte chemoattractant protein-1 (human MCP-1) mRNA accumulated in THP-1 cells 2 h after lipopolysaccharide (LPS) stimulation. DNase I footprinting revealed that LPS stimulation induced protein binding to the two closely located NF-kappaB sites, A1 and A2. By electrophoretic gel mobility shift assay and supershift assay, the binding of (p65)2, c-Rel/p65, p50/p65, and p50/c-Rel to the A2 oligonucleotide probe was detected after LPS stimulation. In contrast, 12-o-tetradecanoylphorbol 13-acetate did not induce a significant amount of MCP-1 mRNA in THP-1 cells 2 h after stimulation, and only p50/p65 bound to the A2 probe. trans-Activity of each NF-kappaB/Rel dimer was investigated by transfecting P19 cells with p65, p50, and/or c-Rel expression vectors, and a luciferase construct containing the enhancer region of the human MCP-1 gene. Expression of recombinant p65 or p65 and c-Rel resulted in elevated luciferase activities, indicating that (p65)2 and c-Rel/p65 had trans-activity. The binding of (p65)2 and/or c-Rel/p65 to the A2 probe was also detected from 12-o-tetradecanoylphorbol 13-acetate-stimulated HeLa, HOS, and A172 cells in which expression of MCP-1 mRNA was elevated. Finally, the role of the A1 site was investigated. Both (p65)2 and c-Rel/p65 bound to the A1 probe by electrophoretic mobility shift assay and a mutation in the A1 or A2 site resulted in a loss of the enhancer activity. These results suggest that the binding of (p65)2 and c-Rel/p65 to the A1 and A2 sites of this gene is important for the tissue- and stimulus-specific transcription of the human MCP-1 gene. PMID: 9388261 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 277: Oncogene. 1997 Dec 11;15(24):2965-74. Rel/NF-kappa B transcription factors and I kappa B inhibitors: evolution from a unique common ancestor. Huguet C, Crepieux P, Laudet V. Laboratoire de regulation des processus invasifs, de l'angiogenese et de l'apoptose, EP560, IFR3 Institut de Biologie de Lille, France. From the sequences of Rel/NF-kappa B and I kappa B proteins, we constructed an alignment of their Rel Homology Domain (RHD) and ankyrin repeat domain. Using this alignment, we performed tree reconstruction with both distance matrix and parsimony analysis and estimated the branching robustness using bootstrap resampling methods. We defined four subfamilies of Rel/NF-kappa B transcription factors: (i) cRel, RelA, RelB, Dorsal and Dif; (ii) NF-kappa B1 and NF-kappa B2; (iii) Relish and (iv) NF-AT factors, the most divergent members. Subfamilies I and II are clustered together whereas Relish diverged earlier than other Rel/NF-kappa B proteins. Three subfamilies of I kappa B inhibitors were also defined: (i) NF-kappa B1 and NF-kappa B2; (ii) close to subfamily I, the short I kappa B proteins I kappa B alpha, I kappa B beta and Bcl-3; (iii) Relish that diverged earlier than other I kappa B inhibitors. Our definition of groups and subfamilies fits to structural and functional features of the Rel/NF-kappa B and I kappa B proteins. We also showed that ankyrin repeats of NF-kappa B1, NF-kappa B2 and Relish are short I kappa B-specific ankyrin motifs. These proteins defining a link between Rel/NF-kappa B and I kappa B families, we propose that all these factors evolved from a common ancestral RHD-ankyrin structure within a unique superfamily, explaining the specificities of interaction between the different Rel/NF-kappa B dimers and the various I kappa B inhibitors. PMID: 9416840 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 278: Mol Cell Biol. 1997 Dec;17(12):7375-85. I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain. Crepieux P, Kwon H, Leclerc N, Spencer W, Richard S, Lin R, Hiscott J. Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Department of Medicine, McGill University, Montreal, Que., Canada. The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation. PMID: 9372968 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 279: Kidney Int. 1997 Oct;52(4):926-33. Rapid communication. Enalapril decreases nuclear factor kappa B activation in the kidney with ureteral obstruction. Morrissey JJ, Klahr S. Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. The transcription factor nuclear factor kappa B (NF-kappa B) controls a number of genes associated with tissue inflammation and has been shown to be activated in the kidney with ureteral obstruction. In this investigation, we further explored NF-kappa B activation in the kidney cortex of rats with unilateral ureteral obstruction. Electrophoretic mobility shift assays combined with antibody supershift/depletion demonstrated that NF-kappa B subunits p50, p52, c-rel, p65 (RelA) and RelB were all activated. Immunocytochemical analysis using an antibody to the p50 subunit demonstrated activation occurring predominantly in nuclei of tubular cells. Treatment of animals with unilateral ureteral obstruction with an oral ACE inhibitor significantly decreased NF-kappa B activation. This suggests that the antiinflammatory effect of ACE inhibitors in renal disease is in part due to a blunting of NF-kappa B activation. PMID: 9328931 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 280: Am J Pathol. 1997 Oct;151(4):891-6. Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes. Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE. Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA. Recently we have demonstrated that inhibition of the nuclear factor (NF)-kappa B/Rel family of transcription factors induces apoptosis of B cells. Interestingly, mice lacking the relA gene encoding the p65 subunit of NF-kappa B exhibit embryonic lethality at days 15 to 16 of gestation, accompanied by massive destruction of liver via apoptosis. To determine whether p65 protein plays a direct role in hepatocyte survival, we employed a nontransformed murine hepatocyte (NMH) cell line, which maintains to a high degree the differentiated hepatocyte phenotype. Exponentially growing NMH cells were found to possess a constitutive level of functional classical (p50/p65) NF-kappa B as assayed by electrophoretic mobility shift analysis, antibody supershift, and transient transfection assays. Treatment of NMH cells with the proteasome inhibitor lactacystin, which prevents degradation of the NF-kappa B inhibitor proteins I kappa B, induced apoptosis. Direct inhibition of the endogenous NF-kappa B activity by microinjection of NMH cells with purified specific inhibitor I kappa B-alpha-glutathione-S-transferase fusion protein or an antibody against p65 protein induced apoptosis. These findings suggest that expression of NF-kappa B/Rel activity in murine hepatocytes acts directly to promote survival of these cells and suggest that apoptosis observed in hepatocytes of mice lacking relA is a direct effect of p65 deficiency. PMID: 9327720 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 281: J Virol. 1997 Nov;71(11):8657-65. Divergent transcriptional regulation among expanding human immunodeficiency virus type 1 subtypes. Montano MA, Novitsky VA, Blackard JT, Cho NL, Katzenstein DA, Essex M. Harvard AIDS Institute, Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA. The current AIDS pandemic represents the uneven spread of multiple genetically related subtypes (A to J) of human immunodeficiency virus type 1 (HIV-1). Notably, HIV-1 E in southeast Asia and HIV-1 C in sub-Saharan Africa are expanding faster and are likely of greater global significance than the HIV-1 B subtype prevalent in the United States and Europe. While many studies have focused on genetic variation among structural genes, we chose to conduct a comparative analysis of the long terminal repeats of HIV-1 E and HIV-1 C isolates and report subtype-specific differences in enhancer copy numbers and sequences, as well as divergent activation in response to the cellular transcriptional activators Rel-p65 and NFATc and viral Tat. This study is the first to identify functional distinctions in promoter architecture between HIV-1 subtypes and raises the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. Divergent transcriptional regulation may explain some of the epidemiologically observed differences in transmission and pathogenesis and underscores the need for further comparative analysis of HIV-1 regulation. PMID: 9343223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 282: Cell Growth Differ. 1997 Oct;8(10):1049-59. Nuclear factor-kappaB/Rel blocks transforming growth factor beta1-induced apoptosis of murine hepatocyte cell lines. Arsura M, FitzGerald MJ, Fausto N, Sonenshein GE. Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118-2394, USA. Treatment of hepatocytes with transforming growth factor beta1 (TGF-beta1) induces growth arrest, which is followed by extensive cell death by apoptosis. Previously, we found that TGF-beta1 down-modulates nuclear factor (NF)-kappaB/Rel activity in murine B cell lymphomas, inducing apoptosis. Furthermore, p65 (RelA)-deficient mice died during gestation due to apoptosis of liver cells. Here we have explored the effects of TGF-beta1 on hepatocytes, using two untransformed murine hepatocyte cell lines, AML-12 and NMH, which constitutively express classical NF-kappaB. TGF-beta1 treatment caused increased NF-kappaB binding that was followed by a dramatic decrease in NF-kappaB levels that preceded apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TGF-beta1. The down-regulation in NF-kappaB activity correlated with elevated IkappaB-alpha expression due to hypophosphorylation and increased IkappaB-alpha protein stability. Thus, NF-kappaB factor expression acts directly to promote liver cell survival. Furthermore, these findings characterize a novel signaling pathway for TGF-beta1 in epithelial cells involving down-regulation of NF-kappaB/Rel factors activity through posttranslational modification of IkappaB-alpha protein. PMID: 9342183 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 283: Mol Cell Biol. 1997 Oct;17(10):6184-90. A new member of the I kappaB protein family, I kappaB epsilon, inhibits RelA (p65)-mediated NF-kappaB transcription. Li Z, Nabel GJ. Department of Internal Medicine, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650, USA. A novel member of the I kappaB family has been identified as a protein that associated with the p50 subunit of NF-kappaB in a yeast two-hybrid screen. Similar to previously known I kappaB proteins, this member, I kappaB epsilon, has six consecutive ankyrin repeats. I kappaB epsilon mRNA is widely expressed in different human tissues, with highest levels in spleen, testis, and lung. I kappaB epsilon interacts with different NF-kappaB proteins, including p65 (RelA), c-Rel, p50, and p52, in vitro and in vivo and inhibits the DNA-binding activity of both p50-p65 and p50-c-Rel complexes effectively. Endogenous and transfected NF-kappaB (RelA-dependent) transcriptional activation is inhibited by I kappaB epsilon. I kappaB epsilon mRNA is expressed at different levels in specific cell types and is synthesized constitutively in transformed B-cell lines. It also displays differential induction in response to tumor necrosis factor alpha, interleukin-1, or phorbol ester stimulation compared to I kappaB alpha in non-B-cell lines. Therefore, I kappaB epsilon represents a novel I kappaB family member which provides an alternative mechanism for regulation of NF-kappaB-dependent transcription. PMID: 9315679 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 284: J Biol Chem. 1997 Aug 29;272(35):22278-84. Distinct domains of the RelA NF-kappaB subunit are required for negative cross-talk and direct interaction with the glucocorticoid receptor. Wissink S, van Heerde EC, Schmitz ML, Kalkhoven E, van der Burg B, Baeuerle PA, van der Saag PT. Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands. The RelA subunit of NF-kappaB and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Here, we show by deletions and point mutations that both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA was found to associate with the glucocorticoid receptor in vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behaved as transdominant inhibitors of the repressive activity of wild type RelA. Furthermore, we show that the 13 S E1A protein is able to interfere with the transrepressive activity of RelA. We propose that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor. PMID: 9268377 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 285: J Biol Chem. 1997 Aug 29;272(35):21774-83. Temporal and subunit-specific modulations of the Rel/NF-kappaB transcription factors through CD28 costimulation. Kahn-Perles B, Lipcey C, Lecine P, Olive D, Imbert J. Unite de Cancerologie Experimentale, U119 INSERM, 27 boulevard Lei Roure, 13009 Marseille, France. Stimulation of highly purified primary T lymphocytes through CD2 and CD28 adhesion molecules induces a long-term proliferation, dependent on persistent autocrine secretion of interleukin 2 (IL-2), high and prolonged expression of inducible CD25/IL-2 receptor alpha chain (IL-2Ralpha), and secretion of growth factors such as the granulocyte-macrophage colony-stimulating factor (GM-CSF). CD28 costimulation appears to activate cytokine gene expression through conserved kappaB-related CD28 response (CD28RE) or cytokine 1 (CK-1) elements in addition to canonical NF-kappaB-binding sites. In this report, we assess: 1) the evolution of the expression, over an 8-day time period, of the Rel/NF-kappaB family of proteins in costimulated versus TcR/CD3-stimulated primary T cells; 2) the impact of changes on the in vitro occupancy of GM-CSF kappaB and CK-1, as well as IL-2Ralpha kappaB sites; and 3) the differential regulation of newly synthesized p65 and c-Rel by IkappaB proteins. We show that CD2+CD28 stimulation specifically induces, at maximal T cell proliferation phase, sustained nuclear overexpression of NFKB2 p52 and c-Rel subunits which might rely on long-lasting processing of p100 precursor for p52 and increased neosynthesis of c-Rel. This up-regulation correlates with sustained occupancy of GM-CSF kappaB and CK-1 elements by both proteins. Conversely, these subunits do not appear to bind to the IL-2Ralpha kappaB site. Costimulation, but not TcR/CD3 stimulation, appears supported by sustained down-regulation of both IkappaBalpha and -beta regulators. Furthermore, contrary to p65, c-Rel appears to display little affinity for p105, p100 and IkappaBalpha regulators. PMID: 9268307 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 286: J Biol Chem. 1997 Aug 22;272(34):21281-8. The 90-kDa ribosomal S6 kinase (pp90rsk) phosphorylates the N-terminal regulatory domain of IkappaBalpha and stimulates its degradation in vitro. Ghoda L, Lin X, Greene WC. University of Colorado Health Sciences Center, Department of Pharmacology, School of Medicine, Denver, Colorado 80262, USA. Nuclear factor kappaB (NF-kappaB) is a eukaryotic member of the Rel family of transcription factors whose biological activity is post-translationally regulated by its assembly with various ankyrin-rich cytoplasmic inhibitors, including IkappaBalpha. Expression of NF-kappaB in the nucleus occurs after signal-induced phosphorylation, ubiquitination, and proteasome-mediated degradation of IkappaBalpha. The induced proteolysis of IkappaBalpha unmasks the nuclear localization signal within NF-kappaB, allowing its rapid migration into the nucleus, where it activates the transcription of many target genes. At present, the identity of the IkappaBalpha kinase(s) that triggers the first step in IkappaBalpha degradation remains unknown. We have investigated the potential function of the 90-kDa ribosomal S6 kinase, or pp90(rsk), as a signal-inducible IkappaBalpha kinase. pp90(rsk) lies downstream of mitogen-activated protein (MAP) kinase in the well characterized Ras-Raf-MEK-MAP kinase pathway that is induced by various growth factors and phorbol ester. We now show that pp90(rsk), but not pp70(S6K) or MAP kinase, phosphorylates the regulatory N terminus of IkappaBalpha principally on serine 32 and triggers effective IkappaBalpha degradation in vitro. When co-expressed in vivo in COS cells, IkappaBalpha and pp90(rsk) readily assemble into a complex that is immunoprecipitated with antibodies specific for either partner. While phorbol 12-myristate 13-acetate produced rapid activation of pp90(rsk), in vivo, other potent NF-kappaB inducers, including tumor necrosis factor alpha and the Tax transactivator of human T-cell lymphotrophic virus, type I, failed to activate pp90(rsk). These data suggest that more than a single IkappaBalpha kinase exists within the cell and that these IkappaBalpha kinases are differentially activated by different NF-kappaB inducers. PMID: 9261139 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 287: J Immunol. 1997 Aug 1;159(3):1319-27. Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells. McGuire KL, Iacobelli M. Department of Biology and Molecular Biology Institute, San Diego State University, CA 92182, USA. kmcguire@sunstroke.sdsu.edu CD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells. PMID: 9233628 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 288: J Virol. 1997 Aug;71(8):5972-81. VBP and RelA regulate avian leukosis virus long terminal repeat-enhanced transcription in B cells. Curristin SM, Bird KJ, Tubbs RJ, Ruddell A. Department of Microbiology and Immunology and Cancer Center, University of Rochester, School of Medicine and Dentistry, New York 14642, USA. The avian leukosis virus (ALV) long terminal repeat (LTR) contains a compact transcription enhancer that is active in many cell types. A major feature of the enhancer is multiple CCAAT/enhancer element motifs that could be important for the strong transcriptional activity of this unit. The contributions of the three CCAAT/enhancer elements to LTR function were examined in B cells, as this cell type is targeted for ALV tumor induction following integration of LTR sequences next to the c-myc proto-oncogene. One CCAAT/enhancer element, termed a3, was found to be the most critical for LTR enhancement in transiently transfected B lymphoma cells, while in chicken embryo fibroblasts all three elements contributed equally to enhancement. Gel shift assays demonstrated that vitellogenin gene-binding protein (VBP), a member of the PAR subfamily of C/EBP factors, is a major component of the nuclear proteins binding to the a3 CCAAT/enhancer element. VBP activated transcription through the a3 CCAAT/enhancer element, supporting the idea that VBP is important for LTR enhancement in B cells. A member of the Rel family of proteins was also identified as a component of the a3 protein binding complex in B cells. Gel shift and immunoprecipitation assays indicated that this factor is RelA. Gel shift assays demonstrated that while RelA does not bind directly to the LTR CCAAT/enhancer elements, it does interact with VBP to potentiate VBP DNA binding activity. The synergistic interaction of VBP and RelA increased CCAAT/enhancer element-mediated transcription, indicating that both factors may be important for viral LTR regulation and also for expression of many cellular genes. PMID: 9223487 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 289: Oncogene. 1997 Jul 3;15(1):29-43. Transcriptional and post-transcriptional regulation of kappaB-controlled genes by pp60v-src. Cabannes E, Vives MF, Bedard PA. Department of Biology & Founders Gate, York University, North York, ON, Canada. The CEF-4/9E3 gene is expressed aberrantly in chicken embryo fibroblasts transformed by the Rous sarcoma virus. This aberrant expression is dependent on transcriptional activation and on the stabilization of the CEF-4 mRNA. The characterization of the CEF-4 promoter indicated that three distinct regulatory elements corresponding to an AP-1 binding site, a PRDII/ kappaB domain and a CAAT box are involved in the activation by pp60v-src. Several v-src responsive genes are controlled by AP-1 and members of the Ets family but few appear to be dependent on NF-kappaB. In this study we characterize the expression of genes regulated by NF-kappaB in normal and RSV-transformed CEF. Run-on transcription analysis indicated that pp60v-src induces the transcription of several genes controlled by NF-kappaB but at different levels. While the transcription of CEF-4 was strongly stimulated, that of NF-kappaB1, c-rel, p53 or IkappaB-alpha was activated more modestly by pp60v-src. In addition the CEF-4 mRNA was the only mRNA species to accumulate significantly in transformed CEF. The ectopic expression of RelA or Rel resulted in the stimulation of the transcription of several known targets of NF-kappaB. However, the mRNA for IkappaB-alpha was the only mRNA species to accumulate considerably in the RelA- or Rel-expressing cells. Hence for most kappaB-controlled genes, transcriptional activation was not sufficient to obtain a significant increase in mRNA expression. Likewise, RelA or Rel enhanced the transcription of the CEF-4 gene without a significant accumulation of the CEF-4 mRNA. However, transformation by v-src caused a massive accumulation of the CEF-4 mRNA but not of other mRNA species in the RelA- and Rel-expressing cells. Transient expression assays, run-on transcription and Northern blotting analyses indicated that the effect of pp60v-src on CEF-4 expression was mediated predominantly at the post-transcriptional level in these cells. Therefore transcriptional and post-transcriptional mechanisms determine the restricted pattern of activation of kappaB-controlled genes in RSV-transformed CEF. PMID: 9233775 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 290: Eur J Immunol. 1997 Jul;27(7):1601-9. Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells. Marienfeld R, Neumann M, Chuvpilo S, Escher C, Kneitz B, Avots A, Schimpl A, Serfling E. Department of Molecular Pathology, University of Wurzburg, Germany. The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50. PMID: 9247567 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 291: Tissue Antigens. 1997 Jul;50(1):1-7. Induction of nuclear factor kappa B/Rel nuclear activity in human peripheral blood T lymphocytes by anti-HLA class I monoclonal antibodies. Turco MC, Romano MF, Lamberti A, Petrella A, Bisogni R, Sun SC, Ferrone S, Bonelli P, Cerra M, Venuta S. Department of Biochemistry, University Federico II, Napels. Monoclonal antibodies against either monomorphic or polymorphic determinants of class I antigen induced in PBMC and highly purified T lymphocytes the nuclear activity of NF-kappa B/Rel complexes. These included both p50/p50 and p50/p65 dimers, recognized by specific antibodies in EMSA. The induced complexes were detectable in extracts of cells incubated with anti-class I monoclonal antibody (mAb) for 1.5 h; the induction was maximal at 5 h, persistent at 16 h and no longer observed at 40 h. The mAb failed to induce NF-kappa B/Rel nuclear activity in cells incubated in the presence of 3,4-dichloroisocoumarin, an inhibitor of I kappa B-alpha degradation. Together, these results suggest that class I triggering can induce the activity of NF-kappa B/Rel nuclear activity in peripheral blood T lymphocytes, thereby modulating the expression of genes regulated by these transcription factors. PMID: 9243748 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 292: J Neuroimmunol. 1997 Jul;77(1):51-6. Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia. Bonaiuto C, McDonald PP, Rossi F, Cassatella MA. Department of General Pathology, University of Verona, Italy. An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS. PMID: 9209268 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 293: J Virol. 1997 Jul;71(7):5692-5. Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB-binding sites of the interleukin-8 gene. Murayama T, Ohara Y, Obuchi M, Khabar KS, Higashi H, Mukaida N, Matsushima K. Department of Microbiology, Kanazawa Medical University, Uchinada, Ishikawa, Japan. Cytomegalovirus (CMV) infection induced interleukin-8 (IL-8) gene transcription in a human monocytic cell line, THP-1 cells, leading to IL-8 secretion. The functional analysis of the IL-8 gene revealed that both AP-1- and NF-kappaB factor-binding elements were involved in conferring the responsiveness to CMV. Moreover, electrophoretic mobility shift assays demonstrated that CMV induced the formation of NF-kappaB and AP-1 complexes. These results suggest that CMV activates these transcriptional factors, resulting in IL-8 gene expression. PMID: 9188651 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 294: Nucleic Acids Res. 1997 Jul 1;25(13):2648-56. Formation of a G-tetrad and higher order structures correlates with biological activity of the RelA (NF-kappaB p65) 'antisense' oligodeoxynucleotide. Benimetskaya L, Berton M, Kolbanovsky A, Benimetsky S, Stein CA. Department of Medicine, Columbia University, College of Physicians and Surgeons, 630 West 168 Street, New York, NY 10032, USA. We have examined the behavior of the phosphorothioate antisense Rel A (NF-kappaB p65) oligodeoxynucleotide (oligo) and related molecules. Because of the presence of a G-tetrad near its 5'terminus, this molecule is capable of forming tetraplexes and other higher order structures in a temperature and time dependent manner. The G-tetrad in the phosphodiester congener is protected from methylation by dimethylsulfate when the oligomer is 3'-phosphorylated. However, this protection is completely lost when it is 5'phosphorylated, indicating that the formation of at least some higher order structures has been blocked. In addition, we also prevented tetraplex formation by substitution of 7-deazaguanosine (7-DG) for guanosine at several positions within and outside of the tetrad. This substitution retains Watson-Crick base pair hybridization but prevents Hoogsteen base-pair interactions. When murine K-Balb cells were treated with 20microM antisense RelA oligo, complete blockade of nuclear translocation of RelA was observed. However, this effect was virtually entirely abrogated in most cases by 7-DG substitution within the tetrad, but retained when the substitution was made 3' to the tetrad. The AS RelA-induced downregulation of Sp-1 activity behaved similarly after 7-DG substitution. Thus, the parent phosphorothioate AS RelA molecule cannot be a Watson-Crick antisense agent. However, these conclusions cannot be extrapolated to other G-tetrad containing oligomers and each must be evaluated individually. PMID: 9185577 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 295: J Biol Chem. 1997 Jun 20;272(25):15928-35. Flanking sequences for the human intercellular adhesion molecule-1 NF-kappaB response element are necessary for tumor necrosis factor alpha-induced gene expression. Paxton LL, Li LJ, Secor V, Duff JL, Naik SM, Shibagaki N, Caughman SW. Emory Skin Diseases Research Core Center, Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. The regulated expression of intercellular adhesion molecule-1 (ICAM-1) by cytokines such as tumor necrosis factor alpha (TNF-alpha) plays an important role in inflammation and immune responses. Induction of ICAM-1 gene transcription by TNF-alpha has previously been shown to be dependent upon a region of the ICAM-1 5'-flanking sequences that contains a modified kappaB site. We demonstrate here that this modified kappaB site alone is insufficient for induction of transcription by TNF-alpha. Site-directed mutagenesis of both the kappaB site and specific flanking nucleotides demonstrates that both the specific 5'- and 3'-flanking sequences and the modified kappaB site are necessary for TNF-alpha induction. Further, site-directed mutagenesis of this modified kappaB site to a consensus kappaB site allows it to mediate transcriptional activation in response to TNF-alpha, even in the absence of specific flanking sequences. Transcription through this minimal ICAM-1 TNF-alpha-responsive region can be driven by co-expression of p65, and the minimal response element interacts with p65 and p50 in supershift mobility shift assays. However, when in vitro transcription/translation products for the Rel proteins are used in an electrophoretic mobility shift assay, only p65 is capable of binding the minimal response element while both p50 and p65 bind a consensus kappaB oligonucleotide. Additionally, in the absence of the specific flanking nucleotides, the ICAM-1 kappaB site is incapable of DNA-protein complex formation in both electrophoretic mobility shift assay and UV cross-linking/SDS-polyacrylamide gel electrophoresis analysis. These results demonstrate the requirement for specific flanking sequences surrounding a kappaB binding site for functional transcription factor binding and transactivation and TNF-alpha-mediated induction of ICAM-1. PMID: 9188493 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 296: J Biol Chem. 1997 Jun 20;272(25):15817-24. The nuclear factor kappa-B signaling pathway participates in dysregulation of vascular smooth muscle cells in vitro and in human atherosclerosis. Bourcier T, Sukhova G, Libby P. Vascular Medicine and Atherosclerosis Unit, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. In the lesions of atherosclerosis, vascular smooth muscle cells (SMC) display many functions characteristic of cytokine activation that likely contribute importantly to ongoing inflammation during human atherogenesis. The transcription factor nuclear factor kappa-B (NFkappaB) often mediates the effects of cytokines on target cells, but the identity of Rel family members important in human SMC activation remains uncertain. In vitro, human SMC express multiple Rel family members. Of these, dimers of p65 and p50, but not a putative SMC-Rel, comprise basal and inducible NFkappaB binding activities. SMC express two inhibitor proteins IkappaBbeta and IkappaBalpha. Interleukin-1beta stimulation caused transient loss of IkappaBalpha and a sustained decrease of IkappaBbeta that correlated with increased and persistent levels of p65/p50 protein and binding activity in the nucleus. SMC cultured under serum-free conditions displayed little NFkappaB activity, but addition of serum or platelet-derived growth factor did activate NFkappaB. In situ analyses showed no evidence for basal NFkappaB activity in SMC in vivo as nonatherosclerotic arteries did not contain nuclear p65 or p50 protein. However, the nuclei of intimal SMC within human atheroma did contain both Rel proteins. We conclude that (i) dimers of p65 and p50, but not SMC-Rel, comprise NFkappaB complexes in human SMC; (ii) stimulatory components in serum activate NFkappaB and likely account for previously reported "constitutive" NFkappaB activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFkappaB activation in SMC because of sustained decreases in the inhibitory subunit IkappaB-beta. PMID: 9188479 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 297: Nucleic Acids Res. 1997 Jun 15;25(12):2424-9. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site. Galien R, Garcia T. Roussel Uclaf, 102 route de Noisy, 93235 Romainville Cedex, France. galien@mac.rousseluclaf.fr Interleukin-6 (IL-6) is a multifunctional cytokine thought to be a key factor in post-menopausal osteoporosis, given its ability to induce osteoclast maturation and its down regulation by estrogens. We have previously shown that the effects of TNFalphaand estradiol on the human IL-6 promoter were dependent on a region of the promoter containing a C/EBP site and a NF-kappaB site. To define the molecular mode of action of estrogens, we performed gel shift assays with this DNA fragment as a probe, and nuclear extracts from TNFalpha-induced HeLa, MCF7 and Saos2 cells. Several induced complexes specifically bound the probe. The use of various competitor DNA suggested that most of the complexes detected contained NF-kappaB factors, and that C/EBP site binding factors were important for the overall binding to the probe. Addition of in vitro translated human estrogen receptor (hER) impaired the binding of three complexes in HeLa cells and two complexes in MCF7 and Saos2 cells. Competition experiments suggested that the NF-kappaB site was necessary for the effect of hER. The use of antisera against NF-kappaB and C/EBP proteins showed that the target complexes of hER contained the c-rel proto-oncogene product and to a lesser extent, the RelA protein. Taken together, these data show that hER impairs TNFalphainduction of IL-6 by preventing c-rel and, to a lesser extent, RelA proteins binding to the NF-kappaB site of the IL-6 promoter. PMID: 9171095 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 298: J Exp Med. 1997 Jun 2;185(11):1897-907. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-kappaB. Boothby MR, Mora AL, Scherer DC, Brockman JA, Ballard DW. Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. Members of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets. PMID: 9166419 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 299: Cytokine. 1997 May;9(5):295-9. Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells. Leonardi A, Altomonte M, Maio M, Tell G, Bearz A, Formisano S, Pucillo C. Department of Sciences and Biomedical Technologies, University of Udine, Italy. The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells. Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens. Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens. In contrast, it was found to be TNF-alpha independent in the early time point. Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens. In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity. PMID: 9195127 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 300: J Interferon Cytokine Res. 1997 May;17(5):295-306. Two forms of NF-kappa B1 (p105/p50) in murine macrophages: differential regulation by lipopolysaccharide, interleukin-2, and interferon-gamma. Brown MC, Tomaras GD, Vincenti MP, Taffet SM. Program in Cell and Molecular Biology, SUNY Health Science Center at Syracuse, USA. In macrophages, nuclear factor kappa B (NF-kappa B) has been shown to transactivate the promoters of many cytokines, including tumor necrosis factor-alpha (TNF-alpha). We have used the -510 kappa B binding site from the murine TNF-alpha promoter to assay the induction of NF-kappa B in murine macrophages by various stimuli. A basal level of NF-kappa B activity in murine macrophages was detectable, and this activity was enhanced by treatment of these cells with lipopolysaccharide (LPS) or interleukin-2 (IL-2). Interferon-gamma (IFN-gamma), an important regulator of macrophage gene expression, significantly enhanced NF-kappa B activity and altered the apparent molecular weight of the NF-kappa B1-like proteins in LPS-stimulated and IL-2-stimulated murine macrophages. The NRD (NF-kappa B/Rel/Dorsal) complexes induced by LPS and IFN-gamma were further characterized by addition of antisera to electrophoretic mobility shift assay (EMSA) reaction mixtures. NF-kappa B1/p50 was a component of all complexes, whereas RelA/p65 was present in the IFN-gamma/LPS-stimulated activity. IFN-gamma priming or treatment with LPS for 19 h resulted in an upregulation of the larger species of NF-kappa B1/p50. In addition, regulation of the two pools of NF-kappa B1/p50 by IFN-gamma was confirmed by Western immunoblot analysis of cytosolic and nuclear extracts. This is the first demonstration of the presence of two pools of NF-kappa B1/p50 differentially regulated in response to cytokine activation of macrophages. PMID: 9181468 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 301: Eur J Immunol. 1997 May;27(5):1091-7. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1. Martin T, Cardarelli PM, Parry GC, Felts KA, Cobb RR. Department of Biology, Tanabe Research Laboratories, San Diego, CA 92121, USA. Chemokines are potent mediators of cell migration and activation and therefore play an essential role in early events of inflammation. In conjunction with cell adhesion molecules, chemokines help to localize cells to a specific site and enhance the inflammatory reaction at the site. Clinically, elevated levels of chemokines have been found in a variety of inflammatory diseases. The prototype C-C chemokine is monocyte chemoattractant protein-1 (MCP-1) which is synthesized by variety of cell types including endothelial cells in response to a variety of stimuli. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we investigated the factors involved in cytokine-induced MCP-1 gene expression in human endothelial cells. We present evidence that the nuclear factor (NF)-kappa B-like binding site and the AP-1 binding site located 90 and 68 base pairs upstream of the transcriptional start site, respectively, are required for maximal induction of the human MCP-1 promoter by interleukin-(IL)-1 beta. Site-directed mutagenesis or deletion of the NF-kappa B-like site decreased the cytokine-induced activity of the promoter. Site-directed mutagenesis of the AP-1 binding site also decreased the cytokine-induced activity of the promoter. We show that the NF-kappa B-like site located at-90 in the MCP-1 promoter binds to the p50/p65 heterodimer of the NF-kappa B/Rel family in IL-1 beta-stimulated human endothelial cells. Overexpression of p65 results in the transactivation of the MCP-1 promoter as well. The data presented in this study suggest that cytokine-induced MCP-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1. PMID: 9174597 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 302: Blood. 1997 May 1;89(9):3421-33. Activation of the NF-kappaB pathway by inflammatory stimuli in human neutrophils. McDonald PP, Bald A, Cassatella MA. Department of General Pathology, University of Verona, Italy. Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-kappaB, for inducible expression. We therefore investigated whether NF-kappaB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFkappaB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IkappaB-alpha in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-alpha [TNF-alpha], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-kappaB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-kappaB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IkappaB-alpha degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IkappaB-alpha mRNA transcripts, resulting in the reexpression of the IkappaB-alpha protein. To our knowledge, this constitutes the first indication that NF-kappaB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology. PMID: 9129050 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 303: Mol Cell Biol. 1997 May;17(5):2605-14. Regulation of the interleukin-2 CD28-responsive element by NF-ATp and various NF-kappaB/Rel transcription factors. Maggirwar SB, Harhaj EW, Sun SC. Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, 17033, USA. The CD28 costimulatory signal enhances antigen-mediated induction of interleukin-2 (IL-2) gene transcription through activation of an enhancer termed the CD28-responsive element (CD28RE). Although various nuclear proteins have been shown to bind to CD28RE, their in vivo functions in the regulation of this enhancer remain elusive. In this report, we show that CD28RE binds distinct transcription factors in cells treated with different mitogenic stimuli. Stimulation of the T-cell receptor (TCR) complex in the absence of a CD28 costimulatory signal induces a member of the nuclear factor of the activated T cells, NF-ATp; however, this treatment fails to activate the CD28RE enhancer activity. Significant activation of CD28RE was detected when the cells were treated with both the TCR stimulators and an anti-CD28 monoclonal antibody (anti-CD28), which induces the NF-kappaB/Rel enhancer binding proteins in addition to NF-ATp. The costimulatory activity of anti-CD28 can be further enhanced by a phorbol ester. Kinetic analyses demonstrate that activation of endogenous IL-2 gene transcription is correlated with the binding of CD28RE by NF-ATp and different NF-kappaB/Rel species. Transient-transfection studies reveal that expression of either NF-ATp or the p50-RelA NF-kappaB heterodimer leads to the potent transactivation of both the CD28RE enhancer and the intact IL-2 promoter in mitogen-stimulated cells. Remarkably, coexpression of these two families of enhancer-binding proteins in Jurkat T cells results in the transactivation of the CD28RE enhancer even in the absence of any cellular stimuli. Together, these results suggest that activation of IL-2 gene transcription by the TCR- and CD28-mediated signals involves the interaction of CD28RE with NF-ATp and various NF-kappaB/Rel transcription factors. PMID: 9111330 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 304: J Biol Chem. 1997 Apr 11;272(15):9825-32. Mechanism for biphasic rel A. NF-kappaB1 nuclear translocation in tumor necrosis factor alpha-stimulated hepatocytes. Han Y, Brasier AR. Department of Internal Medicine and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1060, USA. The proinflammatory cytokine, tumor necrosis factor alpha (TNFalpha), is a potent activator of angiotensinogen gene transcription in hepatocytes by activation of latent nuclear factor-kappaB (NF-kappaB) DNA binding activity. In this study, we examine the kinetics of TNFalpha-activated translocation of the 65-kDa (Rel A) and 50-kDa (NF-kappaB1) NF-kappaB subunits mediated by inhibitor (IkappaB) proteolysis in HepG2 hepatoblastoma cells. HepG2 cells express the IkappaB members IkappaBalpha, IkappaBbeta, and IkappaBgamma. In response to TNFalpha, Rel A.NF-kappaB1 translocation and DNA binding activity follows a biphasic profile, with an "early" induction (15-30 min), followed by a nadir to control levels at 60 min, and a "late" induction (>120 min). The early phase of Rel A.NF-kappaB1 translocation depends on simultaneous proteolysis of both IkappaBalpha and IkappaBbeta isoforms; IkappaBgamma is inert to TNFalpha treatment. The 60-min nadir is due to a rapid IkappaBalpha resynthesis that reassociates with Rel A and completely inhibits its DNA binding activity; the 60-min nadir is not observed when IkappaBalpha resynthesis is prevented by cycloheximide treatment. By contrast, selective inhibition of IkappaBbeta proteolysis by pretreatment of HepG2 cells with the peptide aldehyde N-acetyl-Leu-Leu-norleucinal completely blocks the late phase of Rel A.NF-kappaB1 translocation. These studies indicate the presence of inducible and constitutive cytoplasmic NF-kappaB pools in hepatocytes. TNFalpha induces a coordinated proteolysis and resynthesis of IkappaB isoforms to produce dynamic changes in NF-kappaB nuclear abundance. PMID: 9092517 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 305: J Exp Med. 1997 Apr 7;185(7):1359-70. p50-NF-kappaB complexes partially compensate for the absence of RelB: severely increased pathology in p50(-/-)relB(-/-) double-knockout mice. Weih F, Durham SK, Barton DS, Sha WC, Baltimore D, Bravo R. Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA. RelB-deficient mice (relB(-/-)) have a complex phenotype including multiorgan inflammation and hematopoietic abnormalities. To examine whether other NF-kappaB/Rel family members are required for the development of this phenotype or have a compensatory role, we have initiated a program to generate double-mutant mice that are deficient in more than one family member. Here we report the phenotypic changes in relB(-/-) mice that also lack the p50 subunit of NF-kappaB (p50(-/-)). The inflammatory phenotype of p50(-/-)relB(-/-) double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in contrast to relB(-/-) single knockouts, B cells were absent from inflammatory infiltrates. Both p50(-/-) and heterozygous relB(-/+) animals are disease-free. In the absence of the p50, however, relB(-/+) mice (p50(-/-)relB(-/+)) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased kappaB-binding activities of NF-kappaB/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the "classical" p50-RelA-NF-kappaB activity is not required for the development of the inflammatory phenotype. PMID: 9104822 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 306: EMBO J. 1997 Mar 17;16(6):1413-26. I kappa B epsilon, a novel member of the I kappa B family, controls RelA and cRel NF-kappa B activity. Whiteside ST, Epinat JC, Rice NR, Israel A. Unite de Biologie Moleculaire de l'Expression Genique, UMR 0321 CNRSInstitut Pasteur, Paris, France. We have isolated a human cDNA which encodes a novel I kappa B family member using a yeast two-hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF-kappa B. The protein is found in many cell types and its expression is up-regulated following NF-kappa B activation and during myelopoiesis. Consistent with its proposed role as an I kappa B molecule, I kappa B-epsilon is able to inhibit NF-kappa B-directed transactivation via cytoplasmic retention of rel proteins. I kappa B-epsilon translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, I kappa B-epsilon protein is degraded with slow kinetics by a proteasome-dependent mechanism. Similarly to I kappa B-alpha and I kappa B, I kappa B-epsilon contains multiple ankyrin repeats and two conserved serines which are necessary for signal-induced degradation of the molecule. A unique lysine residue located N-terminal of the serines appears to be not strictly required for degradation. Unlike I kappa B- alpha and I kappa B-beta, I kappa B-epsilon does not contain a C-terminal PEST-like sequence. I kappa B-epsilon would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF-kappa B complexes, distinct from those regulated by other I kappa B proteins. PMID: 9135156 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 307: J Immunol. 1997 Mar 15;158(6):2585-91. Nuclear Rel-A and c-Rel protein complexes are differentially distributed within human thymocytes. Feuillard J, Dargemont C, Ferreira V, Tarantino N, Debre P, Raphael M, Korner M. Service of Biological Hematology, Avicenne Hospital, Bobigny, France. Nuclear factor-kappa B (NF-kappa B)/Rel proteins are inducible transcriptional regulators of numerous cellular genes. They are particularly abundant in lymphoid tissues and are thought to be critical for the transcription of genes involved in immune and inflammatory responses. We have reported previously that a nuclear NF-kappa B activity was present in freshly extracted human thymocytes in the absence of in vitro treatment of these cells. In the present report, we identified NF-kappa B proteins extracted from human thymocyte nuclei as being p50/p65 and p50/c-Rel complexes. Immunochemical and immunofluorescent staining of thymus sections using specific Abs allowed visualization of nuclear NF-kappa B proteins in both thymocytes and nonthymocyte cells. This detection suggested a preferential activation of p50/c-Rel in medullary thymocytes, whereas p50/p65 was present in both cortical and medullary regions of human thymus lobules. However, the intensity of p65 labeling was much higher in several thymocytes from the medulla. p65, p50, and c-Rel activities were found in both CD4- and CD8-positive thymocytes. These observations suggest that p65 and c-Rel complexes play distinct roles in gene expression and that both forms of NF-kappa B play critical roles during late stages of the intrathymic maturation of T cells. PMID: 9058790 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 308: Cell Growth Differ. 1997 Mar;8(3):335-42. Regulation of intercellular adhesion molecule-1 gene by tumor necrosis factor-alpha is mediated by the nuclear factor-kappaB heterodimers p65/p65 and p65/c-Rel in the absence of p50. Aoudjit F, Brochu N, Belanger B, Stratowa C, Hiscott J, Audette M. Department of Biochemistry, Laval University, Quebec, Canada. Human intercellular adhesion molecule-1 (ICAM-1) plays an important role in immune responses as the major specific ligand for the beta2-integrins LFA-1 and Mac-1. During the inflammatory process, ICAM-1 expression is stimulated by various proinflammatory cytokines. We have examined the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by tumor necrosis factor-alpha (TNF-alpha) and by the nuclear factor-kappaB (NF-kappaB) family of transcription factors in the Ad5-transformed human embryonal kidney cell line 293. A proximal site (5'-TTGGAAATTCC-3') mapping at position -228 from the ATG and known to mediate TNF-alpha responsiveness in endothelial cells is also critical for TNF-alpha responsiveness in 293 cells. However, unlike endothelial cells, electrophoretic mobility shift assays, using whole-cell extracts prepared from TNF-alpha-treated cells, showed that TNF-alpha induces the formation of a specific kappaB binding complex, mainly composed of NF-kappaB subunits RelA and c-Rel. Electrophoretic mobility shift assays done with 293 cells transfected with p50, p65, or both subunits showed that p50 only has a weak ability to bind the proximal ICAM-1 NF-kappaB site. Another element exhibiting sequence homology with NF-kappaB binding sites and located at position -540 relative to the mRNA cap site was found to be involved in the basal activity of the ICAM-1 promoter, is not required for TNF-alpha responsiveness, and does not bind NF-kappaB subunits. Whereas transactivation of the ICAM-1 promoter by p65 requires the proximal NF-kappaB site, deletion mutant analysis showed that p50 and, to a greater extent, p52 transactivate reporter plasmids lacking NF-kappaB sites, suggesting the presence of other p50/p52 responsive element(s). PMID: 9056676 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 309: J Clin Invest. 1997 Feb 15;99(4):763-72. Inhibition of bovine endothelial cell activation in vitro by regulated expression of a transdominant inhibitor of NF-kappa B. Anrather J, Csizmadia V, Brostjan C, Soares MP, Bach FH, Winkler H. Sandoz Center for Immunobiology, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA. The activation of endothelial cells is a recurrent phenomenon linked to pathologic conditions such as inflammation, chronic arthritis, allo- and xenograft rejection. To inhibit endothelial cell activation we have constructed a transactivation-deficient derivative of the p65/RelA subunit of NF-kappa B, a transcription factor known to be crucial for the induction of adhesion molecules, cytokines and procoagulants in activated endothelial cells. This protein (p65RHD) comprises the Rel homology domain of the RelA subunit, retaining dimerization, DNA binding, and nuclear localization functions, but is deficient in transcriptional activation, and acts as a competitive inhibitor of NF-kappa B. Our data demonstrate that p65RHD is a potent and specific inhibitor of NF-kappa B-mediated induction of a number of genes, such as I kappa B alpha, IL-8, E-selectin, P-selectin, and tissue factor in endothelial cells. Furthermore, tetracycline-inducible expression of p65RHD in stably transfected primary endothelial cells inhibits the induction of gene expression equally well. This regulated system of gene expression provides the basis for a novel therapeutic approach to the pathologic effects of endothelial cell activation, especially in delayed xenograft rejection, by using transgenic animals as organ donors. PMID: 9045881 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 310: Biochem Mol Med. 1997 Feb;60(1):76-9. Exclusion of the nuclear factor-kappa B3 (REL A) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene. Landsvater RM, de Wit MJ, Peterson LF, Sinke RJ, Geurts van Kessel A, Lips CJ, Hoppener JW. Department of Internal Medicine, University Hospital Utrecht, The Netherlands. Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by neoplasia and hyperplasia in specific endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a region of approximately 900 kb on chromosome 11q13. The nuclear factor-kappa B (NF-kappa B) is a transcription factor with pleiotropic expression, which is involved in the regulation of expression of many cellular genes. The p50/p65 heterodimer is the most abundant form of NF-kappa B. The gene encoding the p65 subunit (NF-kappa B3/REL A) was recently localized in the 900-kb MEN 1 region and was considered a good candidate gene for MEN 1. The structure and nucleotide sequence of the NF-kappa B3 coding region in MEN 1 patients were compared with those of non-MEN 1 subjects, to determine the potential role of this gene in MEN 1 tumorigenesis. Southern blot analysis with constitutional DNA from probands of 14 independent MEN 1 families and DNA from four MEN 1 tumor specimens did not reveal any structural abnormality of the NF-kappa B3 gene. Direct sequencing of cDNAs from two affected subjects from 2 different MEN 1 families, as well as nucleotide sequence analysis of exon/intron boundaries in these patients, did not reveal MEN 1-specific point mutations or other small structural aberrations in the NF-kappa B3 gene. These results make it very unlikely that the NF-kappa B3 gene is the gene responsible for the development of MEN 1. PMID: 9066984 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 311: Arthritis Rheum. 1997 Feb;40(2):226-36. Involvement of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Crofford LJ, Tan B, McCarthy CJ, Hla T. University of Michigan, Ann Arbor, USA. OBJECTIVE: To evaluate involvement of the transcription factor nuclear factor kappa B (NF-kappa B) in the increased expression of cyclooxygenase-2 (COX-2) stimulated by interleukin-1 beta (IL-1 beta) in primary rheumatoid synoviocytes. METHODS: We treated early-passage rheumatoid synoviocytes with IL-1 beta and examined the time course of NF-kappa B translocation to the nucleus by Western blot analysis, as well as NF-kappa B binding to the COX-2 promoter/enhancer by electrophoretic mobility shift assay. We correlated the time course of NF-kappa B binding with expression of COX-2 messenger RNA (mRNA) and protein. Synoviocytes were then treated with either sense or antisense phosphorothioate-modified oligonucleotides derived from the transcription start site of the human NF-kappa B p65 RNA. We analyzed NF-kappa B binding to the COX-2 promoter and COX-2 protein levels after these treatments. RESULTS: IL-1 beta rapidly stimulated the translocation of the p65, p50, and c-rel NF-kappa B subunits from the cytoplasm to the nucleus. Electrophoretic mobility shift assay demonstrated binding to 2 NF-kappa B sites within the COX-2 promoter/enhancer, with a time course identical to that of nuclear localization of NF-kappa B. Supershift analysis revealed that binding activity was due primarily to the p65-p50 heterodimer and the p50 homodimer. With appropriate lag time after NF-kappa B binding, COX-2 mRNA and protein were increased. Pretreatment of RA synoviocytes with NF-kappa B p65 antisense oligonucleotides resulted in decreased binding to the COX-2 promoter and decreased COX-2 protein expression. CONCLUSION: These data demonstrate that signaling via the NF-kappa B pathway is involved in regulation of COX-2 expression induced by IL-1 beta in RA synoviocytes. PMID: 9041934 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 312: Science. 1997 Jan 24;275(5299):523-7. Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ. Howard Hughes Medical Institute, University of Michigan Medical Center, 4520 MSRB I, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA. The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacted with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibited p300-associated cyclin E-Cdk2 activity, stimulated kappaB-dependent gene expression, which was also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression. PMID: 8999795 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 313: J Biol Chem. 1997 Jan 3;272(1):601-8. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. Schmedtje JF Jr, Ji YS, Liu WL, DuBois RN, Runge MS. Sealy Center for Molecular Cardiology, Department of Medicine, The University of Texas Medical Branch, Galveston 77555-1064, USA. john@schmedtje.utmb.edu The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells. PMID: 8995303 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 314: J Biol Chem. 1996 Dec 6;271(49):31496-501. Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation. Roshak AK, Jackson JR, McGough K, Chabot-Fletcher M, Mochan E, Marshall LA. SmithKline Beecham Pharmaceuticals, SmithKline Beecham Pharmaceuticals, Immunopharmacology, UW2532, King of Prussia, Pennsylvania 19406, USA. Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation. PMID: 8940164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 315: Immunity. 1996 Dec;5(6):563-74. Corepression of RelA and c-rel inhibits immunoglobulin kappa gene transcription and rearrangement in precursor B lymphocytes. Scherer DC, Brockman JA, Bendall HH, Zhang GM, Ballard DW, Oltz EM. Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville Tennessee 37232, USA. Multiple members of the NF-kappa B/Rel protein family are induced during B cell differentiation and have been implicated in transcriptional activation of the immunoglobulin kappa (Ig kappa) locus. Despite these findings, normal numbers of Ig kappa + B lymphocytes are produced by mice bearing targeted mutations in individual NF-kappa B/Rel genes. In the present study, precursor B lymphocytes were engineered to express a trans-dominant form of I kappa B alpha that simultaneously impairs the c-Rel and RelA transactivating subunits of NF-kappa B. This dual block in NF-kappa B/Rel signaling led to potent inhibition of germline Ig kappa transcription and rearrangement, whereas recombinase activity was unaffected. These findings suggest that c-Rel and RelA serve compensatory functional roles in the developmental mechanisms that govern Ig kappa gene assembly. PMID: 8986716 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 316: Mol Cell Biol. 1996 Dec;16(12):6736-43. CD28 mediates a potent costimulatory signal for rapid degradation of IkappaBbeta which is associated with accelerated activation of various NF-kappaB/Rel heterodimers. Harhaj EW, Maggirwar SB, Good L, Sun SC. Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey 17033, USA. Optimal activation of T cells requires at least two signals delivered by the T-cell receptor complex and costimulatory molecules such as CD28. The CD28 signaling participates in the transcription of the interleukin-2 gene through activation of an enhancer termed the CD28-responsive element (CD28RE). Stimulation of CD28 enhances mitogen-mediated induction of CD28RE-binding proteins including members of the NF-kappaB/Rel transcription factor family, although the underlying mechanism remains elusive. In this report, we show that CD28 costimulation leads to biphasic induction of NF-kappaB/Rel heterodimers, including early-phase induction of p50/RelA and c-Rel/RelA and late-phase induction of p50/c-Rel. Interestingly, activation of these NF-kappaB/Rel complexes by the CD28 signal is associated with the rapid degradation of both IkappaBalpha and IkappaBbeta, two major cytoplasmic inhibitors of NF-kappaB/Rel. Although IkappaBalpha degradation can be induced by phorbol ester alone, degradation of IkappaBbeta is largely dependent on the CD28 costimulatory signal. We further demonstrate that CD28-mediated transactivation of the CD28RE enhancer is potently inhibited by an N-terminal truncation mutant of IkappaBbeta that is incapable of responding to the degradation signals. Together, these results suggest that the CD28 costimulatory signal augments activation of NF-kappaB/Rel by promoting degradation of IkappaBbeta as well as enhancing degradation of IkappaBalpha and that induction of NF-kappaB/Rel serves as an essential step in the signal-mediated activation of the CD28RE enhancer. PMID: 8943328 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 317: J Immunol. 1996 Nov 15;157(10):4442-50. Identification of a promoter element that regulates tissue-specific expression of the human CD80 (B7.1) gene. Fong TC, Wu Y, Kipps TJ. Department of Medicine, University of California at San Diego Cancer Center, La Jolla 92093, USA. We isolated the promoter region of the gene encoding human CD80 to examine for elements responsible for the regulated expression of this important costimulatory molecule. Using CAT reporter constructs containing a heterologous general enhancer, we demonstrate that the CD80 promoter is active in CD80-expressing Raji cells, but has no significant activity in Jurkat cells that are CD80 negative. Transcriptional activity in Raji increases as the promoter is truncated from nucleotide position -906 to -84. However, truncation of this promoter to -41 significantly decreases its activity. Within this region is one stretch of DNA that is protected in DNase I footprint analysis and that shows some sequence similarity to the NF-kappaB element. Site-specific mutation of the 5' purine-rich portion of this element (B7-RE, or B7 regulatory element) abrogates expression. Nuclear extracts prepared from Raji, or from leukemic cells induced to express CD80, form a distinct complex(es) with B7-RE in electromobility shift assays. Moreover, a consensus NF-kappaB oligonucleotide can compete with B7-RE for nuclear extract binding. However, no super-shifted bands are observed when extracts are preincubated with Abs to p50, p65, or other Rel proteins. Moreover, we find that recombinant p49 (RelB), p50, p65 (RelA), or p49/p65 heterodimers do not bind B7-RE in vitro. These data indicate that B7-RE may help govern expression of genes independent of a tissue-specific enhancer and that this element is bound by nuclear factor(s) other than those that commonly bind NF-kappaB. PMID: 8906820 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 318: Leuk Lymphoma. 1996 Nov;23(5-6):583-92. Nuclear NF-ATp is a hallmark of unstimulated B cells from B-CLL patients. Schuh K, Avots A, Tony HP, Serfling E, Kneitz C. Institute of Pathology, University of Wurzburg, Germany. B lymphocytes from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL) were analysed for the nuclear presence and DNA binding of a panel of transcription factors which are involved in the gene control of lymphoid cells. The following transcription factors were studied: the Octamer factors Oct-1 and Oct-2, members of the AP-1 factor family, NF-AT factors, in particular NF-ATp, and members of the Rel/NF-kB family. We show that the constitutive nuclear translocation of NF-ATp, a member of the growing family of NF-AT factors, is a hallmark of nonstimulated B cells from CLL patients that distinguishes B-CLL cells from 'normal' B lymphocytes. Constitutive nuclear appearance was also observed for NF-kB2/p52. Constitutive binding of further factor proteins to DNA, such as JunD, c-Fos and FosB, was detected in several patients whereas the localisation and DNA binding of other factors such as c-Jun, RelA/p65 and c-Rel was unaltered. It is remarkable that in B-CLL cells the nuclear appearance and DNA binding of specific transcription factors is dramatically affected whereas other members of the same factor family remained unaltered in these leukemic cells. It remains to be shown which molecular events lead to the specific 'pre-activation', i.e. constitutive nuclear translocation and DNA binding, of these members of NF-AT, NF-kB and AP-1 factor families. PMID: 9031090 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 319: Cell Biol Int. 1996 Nov;20(11):777-9. Production of biologically active human RelA (p65) in baculovirus-infected insect cells. Chabot-Fletcher M, Ho ML, Breton JJ, Hanning CR, Amegadzie BY. Department of Immunopharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA. A recombinant baculovirus was constructed to express a cDNA encoding RelA (p65), a member of the NF-kappa B/Rel family of proteins. Infection of Spodoptera frugiderda insect cells with the recombinant baculovirus resulted in the production of the biologically active protein as measured by immunoblotting using RelA-specific antisera and by electrophoretic mobility shift assays. The recombinant protein bound specifically to an oligonucleotide containing the NF-kappa B consensus motif but not to that containing the unrelated Oct-1 consensus motif. Thus insect cell-derived RelA possess properties similar to the native protein and may be used in physical, biochemical, and pharmacological studies. PMID: 8979371 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 320: Immunity. 1996 Nov;5(5):479-89. High mobility group protein I(Y) is required for function and for c-Rel binding to CD28 response elements within the GM-CSF and IL-2 promoters. Himes SR, Coles LS, Reeves R, Shannon MF. Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia. CD28 response elements (CD28REs) within cytokine promoters are variant NF-kappaB-binding sites and are essential for transcription in response to CD28 receptor activation in T cells. We show that the CK-1 element (CD28RE) within the GM-CSF promoter binds the RelA and c-Rel transcription factors in response to CD28 activation. We further show that the high mobility group protein HMG I(Y) can bind to the CD28REs of both GM-CSF and IL-2 and that this binding is critical for c-Rel, but not RelA, binding. A second NF-kappaB site in the GM-CSF promoter that binds p50 and RelA, but neither c-Rel nor HMG I(Y), failed to respond to CD28 activation. Expression of HMG I or c-Rel antisense RNA inhibited CD28 activation of the IL-2 and GM-CSF promoters, implying that HMG I(Y) enhancement of c-Rel binding plays an important role in the activity of the CD28REs. PMID: 8934574 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 321: J Interferon Cytokine Res. 1996 Oct;16(10):783-98. The kappa B enhancer of the human interleukin-6 promoter is necessary and sufficient to confer an IL-1 beta and TNF-alpha response in transfected human cell lines: requirement for members of the C/EBP family for activity. Merola M, Blanchard B, Tovey MG. Laboratory of Viral Oncology, CNRS, UPR, Villejuif, France. The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa B enhancer and a NFIL-6 (C/EBP beta) binding site, which have been reported to be essential for inducibility of the IL-6 gene. We show that the kappa B element alone is sufficient to confer inducibility on the IL-6 gene in cells treated with either IL-1 beta or TNF-alpha. Gel-retardation analysis of nuclear extracts from IL-1 beta or TNF-alpha-treated cells using specific antibodies has shown that at least five retarded complexes bind to the IL-6 kappa B element in addition to NF-kappa B. Furthermore, apart from p50 (NF-kappa B1) and p65 (RelA), no other members of the Rel family are present in these complexes. Comparative analysis with the kappa B enhancer of the immunoglobulin kappa chain gene shows that three of these complexes bind specifically to the IL-6 kappa B enhancer: a complex of p50/NFIL6, a p65 homodimer, and a non-Rel-related constitutive protein. Finally, transfection experiments, in which NF-kappa B subunits, NFIL-6, and NFIL-6 beta (C/EBP delta), were overexpressed in cells transfected with mutated IL-6 enhancer elements linked to a reporter gene show that interaction between members of the two families of factors is required for activation of the IL-6 gene in the absence of the NFIL-6 binding site. We conclude that the kappa B enhancer of the IL-6 promoter is the IL-1 beta and TNF-alpha responsive element and that its activity is dependent on the direct interaction of NF-kappa B with non-Rel transcription factors. PMID: 8910763 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 322: Eur J Immunol. 1996 Oct;26(10):2376-82. Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes. Lapointe R, Lemieux R, Olivier M, Darveau A. Canadian Red Cross Society, Blood Services, Transfusion Centre of Quebec, Canada. In vitro, human B lymphocytes undergo long-term proliferation when activated through CD40, a protein expressed on their cell surface. The nature of CD40-dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown. In this study, a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements. Protein kinase C (PKC) depletion generated by a long-term phorbol 12 myristate 13-acetate treatment had weak effects on proliferation. Rather, tyrosine phosphorylation was shown to be directly involved in mediating CD40-dependent signals. The use of the protein tyrosine kinase (PTK)-specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR), a promoter largely dependent on the binding of nuclear factor kappa B (NF- kappa B). In contrast, the cAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation. Electrophoretic mobility shift assay (EMSA) and supershift assay using an NF-kappa B binding sequence from the kappa light chain as a probe, revealed that both p65 (RelA) and c-Rel were present in CD40-stimulated B cells. While PKC depletion did not alter the NF-kappa B level, treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-kappa B level. These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes. PMID: 8898948 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 323: Virology. 1996 Oct 1;224(1):214-23. Differential regulation of the HIV-1 LTR by specific NF-kappa B subunits in HSV-1-infected cells. Schafer SL, Hiscott J, Pitha PM. Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA. Previous studies have shown that expression of HIV-1 provirus was enhanced in cells co-infected with the herpes virus and that HSV-1-mediated induction of the HIV-1 provirus expression involved both NF-kappa B-dependent and NF-kappa B-independent mechanisms. Nuclear NF-kappa B complexes could be detected approximately 8 hr after HSV-1 infection. Using NF-kappa B-specific antibodies in a mobility shift assay, we have found that HSV-1 increases binding of p50, p65, and c-rel to the HIV-1 NF-kappa B probe in both Jurkat T cells and NIH/3T3 cells HSV-1 infection also increases transiently the levels of p50 mRNA but an increase in the level of p65 mRNA was not detected. Furthermore, HSV-1 infection induces a rapid degradation of the I kappa B alpha protein. Transfection of HIV-1 LTR CAT into cell lines which overexpressed individual NF-kappa B proteins showed the highest constitutive expression of CAT activity in cells overexpressing p65. Infection with HSV-1 further enhanced the expression of HIV-1 LTR CAT in cell lines producing p52, p100, and c-rel. In contrast, HSV-1 did not significantly enhance the expression of HIV-1 LTR CAT in cell lines overexpressing p105 and 1 kappa B gamma. In the transient expression assay the p65/c-rel heterodimer was the most effective inducer of the HIV-I LTR expression. Thus it appears that p65 plays a limited role in the NF-kappa B-dependent activation of the HIV-1 LTR following HSV-1 infection and that the stimulation is mediated by the p50/p65 and p65/c-rel heterodimers. Thus the magnitude of HIV-1 provirus induction depends on the relative levels of NF-kappa B subunits present in the infected cells. PMID: 8862416 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 324: J Biol Chem. 1996 Sep 27;271(39):24151-6. Mutual transcriptional interference between RelA and androgen receptor. Palvimo JJ, Reinikainen P, Ikonen T, Kallio PJ, Moilanen A, Janne OA. Institute of Biomedicine, Department of Physiology, University of Helsinki, FIN-00014 Helsinki, Finland. Cross-modulation between androgen receptor (AR) and NF-kappaB/Rel proteins was studied using various androgen- and NF-kappaB-regulated reporter genes under transient transfection conditions. In COS-1 cells, elevated expression of RelA (p65) repressed AR-mediated transactivation in a dose-dependent manner, whereas NFkappaB1 (p50), another major member of the NF-kappaB family, did not influence transactivation. The repression of AR appeared to involve the N-terminal region of the protein between residue 297 and the DNA-binding domain. RelA-mediated transrepression could not be overcome by increasing the amount of AR. Transcriptional interference between RelA and AR was mutual in that cotransfected AR was able to attenuate transactivation by RelA in a dose- and steroid-dependent fashion. An excess of RelA was able to rescue the repression to some extent. Immunological analyses of RelA and AR protein levels indicated that transrepression was not due to reciprocal decrease in their amounts. Neither did AR increase the concentration of IkappaBalpha, which can sequester and inactivate RelA. Electrophoretic mobility shift assays using extracts from cotransfected cells and purified recombinant proteins showed that AR and RelA did not significantly influence each other's DNA binding activity. Nevertheless, protein-protein interaction experiments demonstrated a weak association between AR and RelA. Collectively, these data suggest that the mutual repression in intact cells is due to formation of AR-RelA complexes that are held together by another partner or to competition for a coactivator required for transcription. PMID: 8798655 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 325: J Biol Chem. 1996 Sep 13;271(37):22479-86. Regulation of HIV-1 long terminal repeats by interaction of C/EBP(NF-IL6) and NF-kappaB/Rel transcription factors. Ruocco MR, Chen X, Ambrosino C, Dragonetti E, Liu W, Mallardo M, De Falco G, Palmieri C, Franzoso G, Quinto I, Venuta S, Scala G. Department of Clinical and Experimental Medicine, Medical School, University of Reggio Calabria, 88100 Catanzaro, Italy. We report the characterization of a CAAT enhancer-binding protein (C/EBP) (NF-IL6) element encompassing the region from -174 to -166 of the U3 long terminal repeat (LTR) region of HIV-1. This C/EBP cis sequence was found to bind to C/EBPbeta and C/EBPdelta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBPbeta or C/EBPdelta expression plasmids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancement of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP factors. This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappaB enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappaB1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappaB and C/EBP factors. We observed that p50 middle dotC/EBPbeta and p50 middle dotC/EBPdelta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappaB sequences. The physical association of NF-kappaB1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBPbeta or C/EBPdelta produced as glutathione S-transferase fusion proteins. Moreover, p50 middle dotC/EBPbeta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappaB oligonucleotides. By using mutant forms of p50 or C/EBPbeta proteins we found that the transactivation of HIV-1 LTR by p50 middle dotC/EBPbeta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBPbeta. PMID: 8798413 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 326: Leuk Lymphoma. 1996 Sep;23(1-2):43-8. The involvement of the candidate proto-oncogene NFKB2/lyt-10 in lymphoid malignancies. Neri A, Fracchiolla NS, Migliazza A, Trecca D, Lombardi L. Laboratorio di Ematologia Sperimentale e Genetica Molecolare, Universita di Milano, Ospedale Maggiore IRCCS, Milan, Italy. NF-kappa B transcription factors regulate the expression of a variety of genes involved in immune responses and cell growth. In higher vertebrates, the NF-kappa B family encompasses five distinct members. Three NF-kappa B proteins, p65/RelA, RelB, and c-rel/Rel, have high transactivating potential in addition to their DNA binding activity. Two subunits, NF-kappa B1p50 and NF-kappa B2p52, coded respectively by the NFKB1 and NFKB2 genes, may only have DNA binding activity. Moreover, p50 and p52 subunits are translated as precursors, respectively p105 and p100, which can be processed into the mature active forms by the removal of their carboxy-terminal ankyrin domain. The five proteins share a homologous amino-terminal domain (rel domain) involved in DNA binding, dimerization, nuclear transport, and binding of regulatory subunits. All members form homo- and heterodimeric complexes with different DNA binding specificity and transactivating potential. Structural alterations of some members of the NF-kappa B gene family have been observed in lymphoid malignancies. In particular, the NFKB2 gene, localized on chromosome 10q24, represents a candidate proto-oncogene, since it has been found rearranged in certain types of lymphoma and more commonly in cutaneous lymphoma. Molecular analysis indicated that these rearrangements may occur as a consequence of chromosomal translocations or small internal chromosomal deletions. Rearrangements cluster within the carboxy-terminal ankyrin domain of the NFKB2 gene leading to the production of carboxy-terminally truncated proteins which, in some cases, are fused to heterologous protein domains. Experimental data showed that these abnormal proteins are constitutively localized in the nucleus, have lost the transcriptional repressor functions typical of normal NF-kappa B2p52 and may be capable of transactivation activity. These findings suggest that abnormal NFKB2 proteins may contribute to lymphomagenesis by altering the NF-kappa B system, both quantitatively and qualitatively, and leading to the activation of specific subsets of kappa B-controlled genes. Publication Types: Review Review, Tutorial PMID: 9021684 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 327: J Virol. 1996 Sep;70(9):5944-53. Transcriptional regulation of human polyomavirus JC: evidence for a functional interaction between RelA (p65) and the Y-box-binding protein, YB-1. Raj GV, Safak M, MacDonald GH, Khalili K. Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. The transcriptional control region of the human neurotropic polyomavirus JC virus contains a consensus NF-kappa B site which has been shown to enhance both basal and extracellular stimulus-induced levels of transcription of JC promoters. Here, we show that the expression of JC late promoter constructs containing the NF-kappa B site is decreased by cotransfection with the NF-kappa B/rel subunits, p50 and p52, but enhanced by the p65 subunit. However, JC promoter constructs lacking the NF-kappa B site were activated by p52 and p50 and repressed by p65. This antithetical response of the JC promoter mapped specifically to the D domain, which is a target site for the cellular transcription factor, YB-1. Band shift studies indicated that YB-1 and p65 modulate each other's binding to DNA: YB-1 augments the affinity of p65 for the NF-kappa B site, while p65 reduces the binding of YB-1 to the D domain. Results from coimmunoprecipitation followed by Western blot (immunoblot) analysis suggest an in vivo interaction between p65 and YB-1 in glial cells. Functionally, YB-1 appears to act synergistically with p65 to control transcription from the NF-kappa B site. A converse pattern is seen with the D domain, in which YB-1 acts synergistically with p50 and p52 to regulate transcription. p50 and p52 may function as transcriptional activators on the D domain by removing the repressive effect of p65 on YB-1 binding to the D domain. On the basis of these data, we propose a model in which NF-kappa B/rel subunits functionally interact with consensus NF-kappa B sites or YB-1-binding sites, with disparate effects on eukaryotic gene expression. PMID: 8709216 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 328: J Virol. 1996 Aug;70(8):5183-93. Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation. DeLuca C, Roulston A, Koromilas A, Wainberg MA, Hiscott J. Lady Davis Institute for Medical Research, Montreal, Quebec, Canada. Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells. PMID: 8764027 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 329: Braz J Med Biol Res. 1996 Jul;29(7):895-903. RelB, a member of the Rel/NF-kappa B family of transcription factors. Ryseck RP, Weih F, Carrasco D, Bravo R. Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA. RelB, originally identified as an immediate early gene product, is a member of the Rel/NF-kappa B family of transcription factors important for the regulation of genes involved in immune and inflammatory processes. RelB by itself is inactive due to its inability to homodimerize and to bind to kappa B sequences. However, in the presence of the Rel/NF-kappa B proteins p50 or p52, RelB is a potent transactivator. Transcriptional activation domains were identified in the NH2 and COOH termini of RelB separated by the approximately 300 amino acids spanning the Rel homogy domain (RHD). The last 120 amino acids of this domain are necessary for the dimerization of RelB and were analyzed in detail by in vitro mutagenesis. RelB forms complexes with p50 and p52 but not with RelA and c-Rel. In contrast to RelA-containing complexes, RelB-containing complexes are only weakly inhibited in their activity by I kappa B alpha. Furthermore, in lymphoid tissues RelB is not associated with I kappa B alpha. In contrast to other members of the Rel/NF-kappa B family, high expression of RelB is limited to interdigitating dendritic cells. Mice with a targeted disrupted relB locus show phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. Publication Types: Review Review, Tutorial PMID: 9070378 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 330: J Virol. 1996 Jul;70(7):4558-66. Hepatitis B virus HBx protein activates transcription factor NF-kappaB by acting on multiple cytoplasmic inhibitors of rel-related proteins. Su F, Schneider RJ. Department of Biochemistry and Kaplan Cancer Center, New York University School of Medicine, New York 10016, USA. The HBx protein is a small polypeptide encoded by mammalian hepadnaviruses that is essential for viral infectivity and is thought to play a role in development of hepatocellular carcinoma during chronic hepatitis B virus infection. HBx is a transactivator that stimulates Ras signal transduction pathways in the cytoplasm and certain transcription elements in the nucleus. To better understand the activities of HBx protein and its mechanism of action, we have explored the manner by which HBx activates the transcription factor NF-kappaB during transient expression. We show that HBx induces prolonged formation, in a Ras-dependent manner, of transcriptionally active NF-kappaB DNA-binding complexes, which make up the family of Rel-related proteins, p50, p52, RelA, and c-Rel. HBx was found to activate NF-kappaB through two distinct cytoplasmic pathways by acting on both the 37-kDa IkappaBalpha inhibitor and the 105-kappaDa NF-kappaB1 precursor inhibitor protein, known as p105. HBx induces phosphorylation of IkappaBalpha, a three- to fourfold reduction in IKBalpha stability, and concomitant nuclear accumulation of NF-kappaB DNA-binding complexes, similar to that reported for human T-cell leukemia virus type 1 Tax protein. In addition, HBx mediates a striking reduction in cytoplasmic p105 NF-kappaB1 inhibitor and p50 protein levels and release of RelA protein that was sequestered by the p105 inhibitor, concomitant with nuclear accumulation of NF-kappaB complexes. HBx mediated only a slight reduction in the cytoplasmic levels of NF-kappaB2 p100 protein, an additional precursor inhibitor of NF-kappaB, which is thought to be less efficiently processed or less responsive to release of NF-kappaB. No evidence was found for HBx activation of NF-kappaB by targeting acidic sphingomyelinase- controlled pathways. Studies also suggest that stimulation of NF-kappaB by HBx does not involve activation of Ras via the neutral sphingomyelin-ceramide pathway. Thus, HBx protein is shown to activate the NF-kappaB family of Rel-related proteins by acting on two distinct NF-kappaB cytoplasmic inhibitors. PMID: 8676482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 331: Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6059-63. Erratum in: Proc Natl Acad Sci U S A 1996 Sep 3;93(18):9991. An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Aizenman E, Engelberg-Kulka H, Glaser G. Department of Cellular Biochemistry, The Hebrew University, Hadassah Medical School, Jerusalem, Israel. "Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation. PMID: 8650219 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 332: Oncogene. 1996 Jun 6;12(11):2385-92. Inhibition of p105 processing by NF-kappaB proteins in transiently transfected cells. Harhaj EW, Maggirwar SB, Sun SC. Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hersey 17033, USA. Regulation of the transcription factor NF-kappaB involves proteasome-mediated processing of the NF-kappaB1 p105 precursor protein, which generates the p50 subunit of NF-kappaB. The processing of p105 occurs constitutively in vivo but can be markedly enhanced by various cellular activation agents, although the underlying regulatory mechanism is not yet clear. In the present study, we demonstrate that signal-mediated induction of p105 processing in human T cells is associated with de novo synthesis of this precursor protein. Transient transfection studies performed in COS7 cells revealed that the newly synthesized p105 protein appears to be more rapidly processed compared to its accumulated form that is already associated with the processed product p50. Interestingly, the processing rate of p105 is markedly inhibited in cells co-transfected with p50 or other NF-kappaB subunits, including RelA and c-Rel, that physically interact with p105. These findings suggest that the processing of p105 is subject to negative regulation by the various NF-kappaB subunits. We further demonstrate that p105 undergoes degradation in lipopolysaccharide-stimulated human monocytic cells. However, the inducible degradation of p105 is not coupled with the generation of p50. Together, these studies demonstrate that the processing and inducible degradation of p105 are differentially regulated. PMID: 8649779 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 333: Scand J Immunol. 1996 Jun;43(6):640-5. Activation of the protein kinase A increases the DNA-binding and transcriptional activity of c-Rel in T cells. Lahdenpohja N, Henttinen T, Hurme M. Department of Microbiology and Immunology, University of Tampere Medical School, Finland. Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is known to have both negative and positive effects on the activation mechanisms of T lymphocytes. The authors have analysed the effect of increased cAMP on the activation of NF-kappa B transcription factor. This factor controls the expression of several genes (e.g. IL-2 and IL-2 receptor) involved in the activation and proliferation of T cells. The authors found that elevation of intracellular cAMP in Jurkat T leukaemia cells activated with phorbol ester (PDBu)/calcium ionophore (A23187) increased the DNA-binding of NF-kappa B as detected by the electrophoretic mobility shift assay (EMSA). Analysis of the subunit composition of the DNA-binding complex indicated that the amount of c-Rel was enhanced while RelA was decreased. Analysis of the effect of elevated cAMP on the degradation of I kappa B-alpha and I kappa B-beta did not reveal an essential change in degradation kinetics of these inhibitor proteins. The elevation of cAMP did not increase the synthesis of c-Rel, but it enhanced the nuclear localization of this protein. Transfection of Jurkat cells with a plasmid kB/TK10-CAT indicated that the increased DNA-binding of c-Rel containing complexes seen in EMSA was also functional. These data imply that the strong and long-lasting c-Rel nuclear localization and DNA-binding induced by protein kinase A is not due to increased c-Rel synthesis or enhanced degradation of the I kappa B inhibitors. Therefore, a direct phosphorylation of the c-Rel protein is the most plausible explanation for these observations. Taken together, these results suggest that cAMP is able to regulate the expression of NF-kappa B-dependent genes in T cells by modifying the composition and subunit activity of NF-kappa B. PMID: 8658053 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 334: Nature. 1996 May 23;381(6580):328-31. Effects on NF-kappa B1/p105 processing of the interaction between the HTLV-1 transactivator Tax and the proteasome. Rousset R, Desbois C, Bantignies F, Jalinot P. CNRS UMR49, Ecole Normale Superieure de Lyon, France. The viral Tax protein, which is encoded by human T-cell leukaemia virus HTLV-I, activates nuclear translocation of the NF-kappa B/Rel transcription factors and relieves cytoplasmic sequestration of RelA and Rel by heterodimerization with NF-kappa B1/p1O5 (refs 1,2). Proteolytic maturation of this precursor protein is performed by the proteasome complex. Here we show that Tax binds specifically to two subunits of the 20S proteasome, HsN3 and HC9. This interaction is weakened with HsN3 and lost for HC9 when a mutant of Tax is substituted that is selectively defective for NF-kappa B activation. Immunoprecipitation shows that p1O5 binds weakly to HC9 and that this interaction is reinforced by Tax. No bridging function of Tax between p1O5 and HsN3 was observed. From these results, we propose that Tax accelerates the proteolytic maturation of P105 by favouring its anchorage to the proteasome. PMID: 8692272 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 335: Mol Cell Biol. 1996 May;16(5):2341-9. IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice. Klement JF, Rice NR, Car BD, Abbondanzo SJ, Powers GD, Bhatt PH, Chen CH, Rosen CA, Stewart CL. Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110, USA. The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin. PMID: 8628301 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 336: J Virol. 1996 May;70(5):3051-9. Regulation of avian leukosis virus long terminal repeat-enhanced transcription by C/EBP-Rel interactions. Bowers WJ, Baglia LA, Ruddel A. Department of Microbiology and Immunology, University of Rochester, New York 14642, USA. The avian leukosis and sarcoma virus long terminal repeat (LTR) enhancers feature directly repeated CCAAT/enhancer element sequences which are also found in many viral and cellular gene enhancers. While most members of the CCAAT/enhancer element-binding protein (C/EBP) transcription factor family exhibit tissue-restricted expression, there may be ubiquitously expressed C/EBP-like factors that regulate widespread CCAAT/enhancer element-driven transcription. An avian C/EBP-related factor designated Al/EBP was previ- ously shown to bind CCAAT/enhancer elements within the avian leukosis virus (ALV) and Rous sarcoma virus (RSV) LTR enhancers in a pattern identical to that of a B-cell LTR-binding factor (W. J. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). An Al/EBP-specific antiserum recognizes a 40-kDa LTR CCAAT/enhancer element-binding protein purified from avian B lymphoma cells. A1/EBP is widely expressed at the mRNA and protein levels, suggesting that this protein could be important not only in regulating widespread expression of the AIN and RSV retroviruses but also in controlling the expression of other viral and cellular gene enhancers that possess CCAAT/enhancer motifs. We also found that an NF-KB/Rel-related protein is a component of the LTR CCAAT/enhancer element binding complex through its interaction with A1/EBP. At least one of the NF-kappaB family members, p65 (RelA), is capable of activating LTR CCAAT/enhancer element-driven transcription. These findings suggest a role for Rel-related factors in the regulation of AIN or RSV LTR-driven transcription via an interaction with Al/EBP. PMID: 8627783 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 337: J Immunol. 1996 Apr 15;156(8):2956-63. Endotoxin-induced activation of the nuclear transcription factor kappa B and expression of E-selectin messenger RNA in hepatocytes, Kupffer cells, and endothelial cells in vivo. Essani NA, McGuire GM, Manning AM, Jaeschke H. Cardivoscular Pharmacology, Pharmacia and Upjohn, Inc., Kalamazoo, MI 49007, USA. Endotoxin causes neutrophil sequestration in the liver and severe vascular and parenchymal cell injury. Inducible adhesion molecules such as intercellular adhesion molecule-1 and selectins are involved in neutrophil recruitment to an inflammatory site in vivo. The objective of our study was to characterize the translocation of the nuclear factor kappa B (NF-kappa B) from the cytoplasm to the nucleus (NF-kappa B activation) during endotoxemia using the electrophoretic mobility shift assay and to investigate its role in regulation of E-selectin gene transcription in liver cells in vivo. Administration of 0.5 mg/kg Salmonella enteritidis endotoxin to male Fischer rats induced a drastic but transient activation of NF-kappa B at the whole liver level. Major subunits identified were p50 (NF-kappa B1), p65 (RelA), and c-Rel, but not p52 (NF-kappa B2). Isolation of liver cells from control animals induced substantial NF-kappa B activation in Kupffer cells and endothelial cells and minor activation in hepatocytes. One hour after endotoxin treatment in vivo, NF-kappa B was uniformly activated in all isolated cells. At 5 h, NF-kappa B activation was reduced to various degrees in all cell types, with hepatocytes showing only a moderate decrease. Northern blot analysis indicated no E-selectin mRNA in all control cells; however, at 1 h, endotoxin induced E-selectin gene transcription transiently in endothelial cells and in Kupffer cells but not in hepatocytes. These data support the hypothesis that NF-kappa B activation is important for E-selectin gene transcription in hepatic vascular lining cells. However, the fact that hepatic parenchymal cells, despite NF-kappa B activation do not express E- selectin mRNA, suggests that NF-kappa B activation alone is not sufficient for E-selectin gene transcription in vivo. PMID: 8609416 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 338: Hypertension. 1996 Apr;27(4):1009-17. Tumor necrosis factor activates angiotensinogen gene expression by the Rel A transactivator. Brasier AR, Li J, Wimbish KA. Department of Medicine, University of Texas Medical Branch, Galveston 77555-1060, USA. Angiotensinogen encodes the only known precursor of angiotensin II, a critical regulator of the cardiovascular system. Transcriptional control of angiotensinogen in hepatocytes is an important regulator of circulating angiotensinogen concentrations. Angiotensinogen transcription is increased by the inflammatory cytokine tumor necrosis factor (TNF)-alpha by a nuclear factor-kappaB-like protein binding to an inducible enhancer called the acute-phase response element. By gel mobility shift assays, we observe two specific acute-phase response element-binding complexes, C1 and C2. The abundance of C2 is not changed by TNF treatment. In contrast, C1 is faintly detected in untreated cells, and its abundance increases by fivefold after stimulation. We identify the nuclear factor-kappaB subunits in these complexes using subunit-specific antibodies in the gel mobility "supershift" assay. The transcriptionally inert nuclear factor-kappaB DNA-binding subunit NF-kappaB1 is present in both control and stimulated hepatocyte nuclei. Its abundance changes weakly upon TNF stimulation. In contrast, the potent transactivating protein Rel A is not found in unstimulated hepatocyte nuclei and is recruited by TNF-alpha into the C1 DNA-binding complex. Overexpression of Rel A results in acute-phase response element transcription. Cotransfection of a chimeric GAL4-Rel A protein with GAL4 DNA-binding sites is a strategy that allows for selective study of Rel A. The GAL4:Rel A chimera is a TNF-alpha-inducible transactivator. Deletion of the amino-terminal 254 amino acids of Rel A produces a constitutive activator (that is no longer TNF-alpha inducible). The cytokine induction of Rel A, then, is mediated through its amino-terminal 254 amino acids. We conclude that Rel A:NF-kappaB1 is a crucial cytokine-inducible transcription factor complex regulating angiotensinogen gene synthesis in hepatocytes and may be involved in controlling the activity of the renin-angiotensin system. PMID: 8613256 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 339: J Clin Invest. 1996 Apr 1;97(7):1715-22. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Institute of Clinical Chemistry and Pathobiochemistry, Technical University Munich, Germany. Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease. PMID: 8601637 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 340: Oncogene. 1996 Mar 7;12(5):1159-64. The ankyrin repeats but not the PEST-like sequences are required for signal-dependent degradation of IkappaBalpha. Aoki T, Sano Y, Yamamoto T, Inoue JI. Department of Oncology, The Institute of Medical Science, The University of Tokyo, Japan. The nuclear activity of Rel/NFkappaB transcription factors is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called IkappaBalpha. IkappaBalpha is rapidly phosphorylated and degraded in response to the stimulation through tumor necrosis factor alpha (TNFalpha) receptor, interleukin-1 receptor or CD40. To explore the molecular mechanisms of signal-induced depletion of IkappaBalpha, we have delineated the domain in IkappaBalpha that is required for TNFalpha-induced phosphorylation and rapid degradation of IkappaBalpha. In contrast to the previous reports, the PEST-like sequences, which are present in the carboxyl-terminal region of IkappaBalpha, are demonstrated here to be dispensable for TNFalpha-induced degradation but could be required for signal-independent degradation, as in the case of Cactus, Drosophila homologue of IkappaB. Furthermore, the ankyrin repeats, which are essential for forming a complex with Rel and RelA, are required for TNFalpha-induced degradation suggesting that the putative IkappaB protease could interact with IkappaBalpha in complex with RelA or could recognize the structure of ankyrin repeats. Our data also indicate that neither the ankyrin repeats nor the PEST-like sequences, are essential for TNFalpha-induced phosphorylation. PMID: 8649809 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 341: Microbiol Res. 1996 Mar;151(1):99-103. Influence of relA locus on electrophoretic mobility of a stringent/relaxed Escherichia coli K 12 strain pair. Gitter B, Hummel A, Glombitza F. Hans-Knoll-Institut fur Naturstoff-Forschung e. V., Jena, Federal Republic of Germany. Stringent (relA+) and relaxed (rel A-) controlled Escherichia coli cells differ in their regulation of many biochemical pathways such as phospholipid and lipopolysaccharide metabolism (LPS) after amino acid limitation. Because such differences could result in various cell envelopes, cells of stringent controlled E. coli strain CP78 (relA+) and relaxed controlled E. coli strain CP79 (relA) were studied regarding their electrophoretic mobility. The graphs of the mobility distributions of both strains were different: cells of strain CP79 caused secondary peaks in addition to the main peaks whereas the mobility distributions of cells of strain CP78 showed only one maximum. In the pH range from 6.0 to 8.0 the location of the main peaks of cells of strain CP79 were changed to less negative values after induction of relaxed response. In contrast to this the stringent response in strain CP78 caused no change of the mobility distributions. PMID: 8857269 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 342: Mol Endocrinol. 1996 Mar;10(3):252-64. Angiotensinogen gene activation by angiotensin II is mediated by the rel A (nuclear factor-kappaB p65) transcription factor: one mechanism for the renin angiotensin system positive feedback loop in hepatocytes. Li J, Brasier AR. Departments Internal Medicine and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, USA. The renin-angiotensin system controls blood pressure through the enzymatic production of the vasopressor angiotensin II (AII) from the angiotensinogen (AGT) precursor. Intravascular AII production stimulates de novo synthesis of its precursor in a positive feedback loop through increased gene expression. In this study, we investigate the effects of AII on AGT gene expression. At nanomolar concentrations, All activates transcription of the native AGT gene; this region is mapped to the AGT gene multihormone-inducible enhancer (-615 to -470). Within the multihormone-inducible enhancer, site-directed mutations of the acute-phase response element (APRE) that interfere with nuclear factor-kappa B (NF-kappa B) transcription factor binding also abolish All responsiveness. The APRE functions as a rapidly inducible All-inducible enhancer with peak reporter activity detected after a 4-h stimulation; this effect occurs only when the type 1 AII receptor is expressed. All induces sequence-specific NF-KB binding to the APRE in HepG2 nuclear extracts. Moreover, AII infusions of primary rat hepatocyte cultures produces a rapid 4-fold increase in sequence-specific NF-kappa B binding to the APRE. Antibodies against the transcriptional activator subunit, Rel A, quantitatively supershift the nucleoprotein complex, whereas antibodies to other NF-kappa B members do not, demonstrating that Rel A APRE-binding activity is AII-inducible. Transient overexpression of Rel A(1-551) activates the AGT multihormone-inducible enhancer. AII-inducible domains of Rel A were mapped by cotransfecting a chimeric GAL4-Rel A fusion protein with a reporter gene containing GAL4-binding sites. GAL4-Rel A(1-551) was an AII-inducible transactivator. Deletion of the NH(2)-terminal 254 amino acids of Rel A produces a constitutive transactivator, indicating that Rel A is activated by AII in a manner dependent on its NH(2) terminus. These studies define one mechanism for the renin-angiotensin system positive feedback loop in hepatocyctes. PMID: 8833654 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 343: J Bacteriol. 1996 Mar;178(5):1401-11. Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis. Mechold U, Cashel M, Steiner K, Gentry D, Malke H. Institute for Molecular Biology, Jena University, Germany. We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants. PMID: 8631718 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 344: Nucleic Acids Res. 1996 Feb 15;24(4):737-45. Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides. Khaled Z, Benimetskaya L, Zeltser R, Khan T, Sharma HW, Narayanan R, Stein CA. Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA. Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media. PMID: 8604318 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 345: Virology. 1996 Feb 1;216(1):284-7. Differential effects of I kappa B molecules on Tat-mediated transactivation of HIV-1 LTR. Harhaj E, Blaney J, Millhouse S, Sun SC. Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center 17033, USA. The tat gene product of the human immunodeficiency virus type 1 (HIV-1) strongly induces the transcription directed by the viral long terminal repeat (LTR). Tat acts by interacting with a target RNA element located immediately downstream of the initiation site. In addition, the action of Tat appears to be assisted by the upstream DNA enhancer elements, including the binding sites for the NF-kappa B/Rel family of host transcription factors. In the present study, we demonstrate that Tat transactivation of the HIV-1 LTR is markedly inhibited by several cytoplasmic inhibitors of NF-kappa B/Rel, suggesting the critical involvement of these host transcription factors in the function of the viral Tat protein. Furthermore, the various NF-kappa B inhibitors appear to have differential effects on Tat. While I kappa B alpha, I kappa B beta, and p100 potently inhibit Tat-mediated transactivation, p105 fails to inhibit, but even moderately synergizes, the action of Tat. We further demonstrate that the action of these NF-kappa B/Rel inhibitors on Tat correlates with their inhibitory activities on the RelA subunit of NF-kappa B. Finally, we show that a degradation-resistant I kappa B alpha mutant is able to potently inhibit Tat-mediated activation of the HIV-1 LTR in both untreated and tumor necrosis factor alpha-stimulated T cells, thus suggesting that such an I kappa B alpha mutant may serve as a constitutive repressor of HIV-1 LTR. PMID: 8615004 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 346: Oncogene. 1996 Jan 18;12(2):445-9. Overexpression of RelB in transgenic mice does not affect I kappa B alpha levels: differential regulation of RelA and RelB by the inhibitor protein. Weih F, Lira SA, Bravo R. Department of Molecular Oncology, Bristol-Myers Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA. In mouse lymphoid tissues, RelB heterodimers represent the constitutive kappa B-binding activity, whereas RelA and c-Rel complexes most likely are involved in inducible kappa B-binding and gene activation. Our laboratory has previously shown that the potential excess of NF-kappa B activity in transgenic mice overexpressing RelA is counteracted by a dramatic increase in I kappa B alpha, mainly due to its increased stability through association with RelA. As an attempt to elucidate the in vivo mechanisms that lead to the constitutive DNA-binding activity of RelB heterodimers, we have generated mouse lines overexpressing a relB transgene in a position-independent and copy number-dependent manner. Expression of RelB in these transgenic animals is very high in immature thymocytes and restricted to T cell areas in secondary lymphoid tissues. In contrast to the results obtained with RelA-transgenic thymocytes, we demonstrate here that overexpression of RelB results in a dramatic increase in overall kappa B-binding activity. Interestingly, I kappa B alpha protein levels are not altered in the RelB-transgenic animals, indicating that within the same cell type RelA and RelB complexes are differentially regulated by I kappa B alpha. PMID: 8570223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 347: Anticancer Res. 1996 Jan-Feb;16(1):61-9. Transcription factor decoy approach to decipher the role of NF-kappa B in oncogenesis. Sharma HW, Perez JR, Higgins-Sochaski K, Hsiao R, Narayanan R. Division of Oncology, Roche Research Center, Hoffman-La Roche Inc., Nutley, NJ 07110, USA. Antisense inhibition of the RelA subunit of NF-kappa B transcription factor (but not the NFKB1 subunit) causes pronounced inhibition of tumor cell growth in vitro and in vivo. Inhibition of either subunit, however, results in inhibition of the heterodimeric NF-kappa B complex in antisense-treated cells. Either of the subunits of NF-kappa B can form homo- or heterodimers with other members of the Rel oncogene family. In an effort to decipher the role of homo- vs heterodimeric NNF-kappa B in regulating tumor cell growth, we have used a decoy approach to trap these complexes in vivo. Using double-stranded phosphorothioates as a direct in vivo competitor for homo- vs heterodimeric NF-kappa B, we demonstrate that decoys more specific to RelA inhibit growth tumor cell growth in vitro. We demonstrate that RelA, either as a homodimer or a heterodimer with some other members of the Rel family and not the classical NF-kappa B (RelA/NFKB1), is involved in the differential growth control of tumor cells. Our results indicate that such transcription factor decoys can be a non-antisense tool to study the function of DNA-binding transcription factors. PMID: 8615671 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 348: Blood. 1996 Jan 1;87(1):25-9. REL proto-oncogene is frequently amplified in extranodal diffuse large cell lymphoma. Houldsworth J, Mathew S, Rao PH, Dyomina K, Louie DC, Parsa N, Offit K, Chaganti RS. Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Comparative genomic hybridization (CGH) analysis of DNA extracted from a diffuse lymphoma with a large cell component (DLLC) that displayed double minute chromosomes upon conventional karyotypic analysis indicated overt amplification of DNA sequences derived from the 2p13-15 region. Southern blot analysis of this tumor DNA with a cDNA probe for the proto-oncogene REL, previously mapped to 2p14-15, indicated a greater than 35-fold amplification of REL. To determine the incidence of REL amplification and possible clinical or histologic association with DLLC, a panel of 111 tumor DNAs from DLLC specimens was screened for REL amplification by Southern blot analysis. A copy number of > or = 4 was noted in 26 cases (23%). Southern blot analysis of these 26 tumor DNAs with a cDNA probe for TGFA, mapped to 2p13, indicated lack of coamplification except in one case. Another member of the Rel/NF-kappa B family of transcriptional activators, RELA/p65 mapped to 11q13, was amplified in five cases as determined by Southern blot analysis using a cDNA probe. Nineteen of the 26 DLLC (73%) with REL amplification were primary extranodal lymphomas. As a group, the tumors with REL amplification demonstrated an increased frequency of chromosomal aberrations previously associated with tumor progression, suggesting an oncogenic effect of amplified REL in B-lymphoid cells that already contained a transforming genetic lesion. Thus, REL amplification is a frequent event in DLLC, and probably constitutes a progression-associated marker of primary extranodal lymphomas. This study shows the usefulness of the CGH technique in identifying chromosomal regions overrepresented in tumors that can point to amplified genes and may be correlated with clinical features of the disease. PMID: 8547649 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 349: Biochem J. 1996 Jan 1;313 ( Pt 1):39-44. Transcriptional activation of the chicken lysozyme gene by NF-kappa Bp65 (RelA) and c-Rel, but not by NF-kappa Bp50. Phi van L. Institute for Small Animal Research, Federal Research Center for Agriculture, Molecular Genetics Reseach Unit, Celle, Federal Republic of Germany. The lysozyme gene is expressed at a low level in myeloblasts and is progressively activated to constitutively high expression in mature macrophages. The binding activity of the newly defined NF-kappa B/Rel family of transcription factors increases during the terminal differentiation of macrophages. In this study, I show that NF-kappa B/Rel-like proteins bind to the nuclear factor kappa B (kappa B)-like sequence of the lysozyme promoter. These binding activities were induced by treatment of HD11 cells with lipopolysaccharide. Immunomobility shift assays show that c-Rel is possibly a factor in the complexes that bind to the kappa B-like sequence lys kappa B. Binding activity to one of the protein complexes seems to be regulated by phosphorylation. In fact, overexpression of p65 and c-Rel stimulates expression of the chloramphenicol acetyltransferase gene controlled by the lysozyme promoter. Furthermore, co-transfection experiments reveal that the kappa B-like sequence within the lysozyme promoter mediates the transactivation by p65 and c-Rel. These results indicate that the p65 and c-Rel could be components of the protein complexes that bind to the kappa B-like sequence and this binding could contribute to the progressively activated expression of the lysozyme gene during the terminal differentiation of macrophages. PMID: 8546707 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 350: J Biotechnol. 1995 Dec 1;43(2):139-43. Amplification of lambda plasmids in Escherichia coli relA mutants. Wegrzyn G. Department of Molecular Biology, University of Gdansk, Poland. It was previously demonstrated that, contrary to wild-type stringent (rel+) strains of Escherichia coli, in amino acid-starved relaxed (relA) mutants the replication of lambda plasmid proceeds for several hours. The replication leads to amplification of lambda plasmid DNA. Here, the conditions for this amplification have been optimized. The amplification efficiency depends on the temperature as well as on the nature of amino acid starvation, but it is only little or totally not dependent on the pH value of the medium in a range from 6.0 to 8.0. It seems that the most efficient amplification can be achieved by overnight cultivation of E. coli relA arg strain harbouring lambda plasmid at 36-39 degrees C in minimal medium containing Casamino acids. Under these conditions, the copy number of lambda plasmid increases from about 40 to about 300 per cell giving greater than 7-fold amplification. Publication Types: Review Review, Tutorial PMID: 8562019 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 351: Blood. 1995 Dec 1;86(11):4144-52. Salicylates inhibit lipopolysaccharide-induced transcriptional activation of the tissue factor gene in human monocytic cells. Oeth P, Mackman N. Department of Immunology, Scripps Research Institute, La Jolla, CA 92037, USA. Binding of plasma Factor VII/VIIa to the tissue factor (TF) receptor initiates the coagulation protease cascades. TF expression by circulating monocytes is associated with thrombotic and inflammatory complications in a variety of diseases. Transcriptional activation of the human TF gene in monocytic cells exposed to bacterial lipopolysaccharide (LPS) is mediated by binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter. Here, we report that a family of anti-inflammatory agents, known as the salicylates, inhibited LPS induction of TF activity and TF gene transcription in human monocytes and monocytic THP-1 cells at clinically relevant doses. Furthermore, sodium salicylate blocked the LPS-induced proteolytic degradation of I kappa B alpha, which prevented the nuclear translocation of c-Rel/p65 heterodimers. In contrast, two other nonsteroidal anti-inflammatory drugs, ibuprofen and indomethacin, did not inhibit LPS induction of the TF gene. These results indicated that salicylates inhibited LPS induction of TF gene transcription in monocytic cells by preventing nuclear translocation of c-Rel/p65 heterodimers. The clinical benefits of salicylates in the treatment of several diseases, including atherosclerosis and rheumatoid arthritis, may be related to their ability to reduce monocyte gene expression. PMID: 7492771 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 352: Int Immunol. 1995 Nov;7(11):1709-20. Pleiotropic effects of Bcl-2 on transcription factors in T cells: potential role of NF-kappa B p50-p50 for the anti-apoptotic function of Bcl-2. Ivanov VN, Deng G, Podack ER, Malek TR. Department of Microbiology and Immunology, University of Miami School of Medicine, FL 33101, USA. Bcl-2 functions to repress apoptosis by regulation of genes which encode proteins required for programmed cell death and by interference with peroxidative damage. We investigated the interrelationship between expression of bcl-2 and regulation of transcription factor DNA binding activities in the 2B4 T cell hybridoma and IL-2-dependent CTLL T cell line. Over-expression of bcl-2 in 2B4 resulted in enhanced basal levels of activator protein (AP)-1, octamer binding factor (Oct)-1, lymphoid enhancer binding factor (LEF)-1, RelA-p50 and NF-kappa B p50-p50 DNA binding activities. After apoptotic signaling, down-regulation of AP-1, NF-AT and Oct-1 binding activities was observed in control 2B4 and CTLL, whereas suboptimal, but higher, levels of these transcription factors were found in bcl-2-transfected cells, potentially promoting cell survival. Furthermore, after apoptotic signaling, expression of bcl-2 led to differential changes of NF-kappa B levels, resulting in a decrease in RelA-p50 and an increase in NF-kappa B p50-p50, altering the ratio of these DNA binding activities such that now p50-p50 markedly predominated in both 2B4-Bcl-2 and CTLL-Bcl-2. Apoptotic signaling in the presence or absence of Bcl-2 resulted in induction of the RelB-p50 heterodimer in 2B4. The changes in NF-kappa B/Rel levels raise the possibility that this family of transcription factors may play an important role in the regulation of apoptosis. PMID: 8580069 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 353: Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10242-6. Interactions of a Rel protein with its inhibitor. Lehming N, McGuire S, Brickman JM, Ptashne M. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. Cactus, a Drosophila homologue of I kappa B, binds to and inhibits Dorsal, a homologue of the p50 and p65 components of NF-kappa B. We describe experiments in yeast with various Dorsal and Cactus derivatives showing that Cactus blocks the DNA binding and nuclear localization functions of Dorsal. In contrast, Dorsal's transcriptional activating region is functional in the Dorsal-Cactus complex. We identify two Dorsal mutants, Dorsal C233R and Dorsal S234P, that escape Cactus inhibition in vivo, and we show that these mutants fail to interact with Cactus in vitro. From this and data of others, we identify the likely surface of Dorsal that binds Cactus. We also describe a modified PCR mutagenesis procedure, easier to use than conventional methods, that produces a library of high complexity. PMID: 7479760 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 354: Nucleic Acids Res. 1995 Oct 11;23(19):3887-93. Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1. Reuning U, Wilhelm O, Nishiguchi T, Guerrini L, Blasi F, Graeff H, Schmitt M. Frauenklinik, Technischen Universitat Munchen, Klinikum rechts der Isar, Germany. The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis. PMID: 7479032 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 355: J Immunol. 1995 Oct 1;155(7):3593-600. Two structurally distinct kappa B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages. Ohmori Y, Fukumoto S, Hamilton TA. Department of Immunology, Cleveland Clinic Foundation, OH 44195, USA. The mouse KC gene is an alpha-chemokine gene whose transcription is induced in mononuclear phagocytes by LPS. DNA sequences necessary for transcriptional control of KC by LPS were identified in the region flanking the transcription start site. Transient transfection analysis in macrophages using deletion mutants of a 1.5-kb sequence placed in front of the chloramphenicol acetyl transferase (CAT) gene identified an LPS-responsive region between residues -104 and +30. This region contained two kappa B sequence motifs. The first motif (position -70 to -59, kappa beta 1) is highly conserved in all three human GRO genes and in the mouse macrophage inflammatory protein-2 (MIP-2) gene. The second kappa B motif (position -89 to -78, kappa B2) was conserved only between the mouse and the rat KC genes. Consistent with previous reports, the highly conserved kappa B site (kappa B1) was essential for LPS inducibility. Surprisingly, the distal kappa B site (kappa B2) was also necessary for optimal response; mutation of either kappa B site markedly reduced sensitivity to LPS in RAW264.7 cells and to TNF-alpha in NIH 3T3 fibroblasts. Although both kappa B1 and kappa B2 sequences were able to bind members of the Rel homology family, including NF kappa B1 (P50), RelA (65), and c-Rel, the kappa B1 site bound these factors with higher affinity and functioned more effectively than the kappa B2 site in a heterologous promoter. These findings demonstrate that transcriptional control of the KC gene requires cooperation between two kappa B sites and is thus distinct from that of the three human GRO genes and the mouse MIP-2 gene. PMID: 7561058 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 356: Oncogene. 1995 Sep 7;11(5):993-8. Activation of NF-kappa B/Rel by Tax involves degradation of I kappa B alpha and is blocked by a proteasome inhibitor. Maggirwar SB, Harhaj E, Sun SC. Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey 17033, USA. The tax gene product of the human T-cell leukemia virus type I (HTLV-I) induces the nuclear expression and biological function of the NF-kappa B/Rel family of host transcription factors although the underlying mechanism remains unclear. In the present study, we demonstrate that Tax-mediated activation of NF-kappa B/Rel can be inhibited by a proteasome inhibitor, suggesting the involvement of proteolytic reactions in this Tax-specific activation pathway. Transient transfection and reporter gene assays have revealed that Tax overrides the inhibitory function of I kappa B alpha in both F9 embryonal cells and Jurkat T cells. Moreover, Tax-mediated inactivation of I kappa B alpha requires a 16 amino acid sequence element located at the N-terminal region (amino acid 21-36) of I kappa B alpha, which is also required for tumor necrosis factor alpha-induced degradation of this inhibitory protein. We further demonstrate that the proteasome inhibitor also blocks the degradation of I kappa B alpha observed in HTLV-I-infected T cells. Interestingly, inhibition of I kappa B alpha degradation in these cells led to the accumulation of a phosphorylated form of I kappa B alpha. Together, these studies suggest that Tax activation of NF-kappa B/Rel may involve induction of phosphorylation and subsequent proteasome-mediated degradation of the inhibitor I kappa B alpha. PMID: 7675460 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 357: Oncogene. 1995 Sep 7;11(5):811-23. I kappa B-alpha-mediated inhibition of nuclear transport and DNA-binding by Rel proteins are separable functions: phosphorylation of C-terminal serine residues of I kappa B-alpha is specifically required for inhibition of DNA-binding. Sachdev S, Rottjakob EM, Diehl JA, Hannink M. Department of Biochemistry, University of Missouri-Columbia 65211, USA. I kappa B-alpha inhibits both DNA-binding and nuclear translocation of dimeric Rel complexes that contain either the RelA or c-Rel proteins. These inhibitory functions of I kappa B-alpha proteins are regulated by both constitutive and inducible phosphorylation. We have mapped the constitutive phosphorylation sites of p40, the avian I kappa B-alpha protein, to a C-terminal acidic serine-rich region that contains four serine residues. Deletions or point mutations that significantly alter the overall negatively charged character of this region abolish association of p40 with Rel proteins in vitro. Serine-to-alanine amino acid substitutions in this region modulate the association of p40 with Rel proteins in vitro and abolish p40-mediated inhibition of DNA-binding by c-Rel. Substitution of aspartic acid residues for the phosphorylated serine residues has no effect on p40-mediated inhibition of DNA-binding. In contrast, the C-terminal acidic serine-rich region is not required for p40-mediated inhibition of nuclear translocation of Rel proteins. Our results demonstrate that p40-mediated inhibition of nuclear translocation and inhibition of DNA-binding by Rel proteins are separable functions. Our results suggest that the phosphorylation status of C-terminal serine residues of I kappa B-alpha proteins will be an important aspect of the autoregulatory feedback loop that enforces temporal control of Rel-regulated gene expression. PMID: 7675442 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 358: Anticancer Res. 1995 Sep-Oct;15(5B):1857-67. A DNA motif present in alpha V integrin promoter exhibits dual binding preference to distinct transcription factors. Sharma HW, Higgins-Sochaski K, Perez JR, Narayanan R. Division of Oncology, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ 07110, USA. Antisense inhibition of the RelA subunit but not the NFKB1 subunit of NK-kappa B transcription factor results in a block of cellular adhesion and inhibition of tumor cell growth in vitro and in vivo. Studies aimed at dissecting the molecular mechanism of antisense relA action led to our identification of a kappa B-like motif present in aV integrin promoter. The alpha V/kappa B motif is closely related to RelA/c-Rel-binding sequences, such as 65-2 and TF-1. However, unlike these two kappa Blike motifs, the alpha V/kappa B motif detected a nuclear Sp1 activity distinct from kappa B activity, which was subsequently confirmed to be derived from Sp1. In comparison to the conventional GC box-containing Sp1 motif, the alpha V/kappa B motif also binds in vitro to c-Rel and RelA but not to NFKB1. Antisense inhibition of RelA inhibited the alpha V/kappa B activity. Direct in vivo competition of alpha V/kappa B-binding activity by a decoy approach also resulted in inhibition of alpha V/kappa B activity in intact cells. A variant of the alpha V/kappa B motif was found to retain the dual ability to detect Sp1 and the NF-kappa B complex in the nuclear and cytoplasmic extracts. Such dual interacting ability of a DNA motif offers yet another way of gene regulation in vivo and hence can affect cellular growth. Our results identify alpha V integrin as one of the molecular targets for relA/NF-kappa B and may explain growth inhibition by antisense relA. PMID: 8572570 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 359: Cell Growth Differ. 1995 Aug;6(8):965-76. Retrovirus-mediated transfer of nuclear factor-kappa B subunit genes modulates I kappa B alpha and interferon beta expression. Bitar R, Beauparlant P, Lin R, Pitha P, Hiscott J. Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada. Nuclear factor (NF)-kappa B proteins regulate the transcription of numerous genes involved in the immune response, transcription control, and viral pathogenesis. To examine the effect of ectopic expression of NF-kappa B proteins on DNA-binding activity and gene expression, individual NF-kappa B subunit genes were introduced into NIH 3T3 cells via retrovirus-mediated gene transfer. Expression of NF-kappa B subunits RelA (p65), NF-kappa B1 (p105), NF-kappa B2 (p100), and c-Rel increased the basal level of nuclear NF-kappa B DNA binding in NIH 3T3 cells, whereas expression of delta RelA (p65 delta) and NF-kappa B2 (p52) subunits did not affect basal level activity. Tumor necrosis factor-alpha treatment of the NF-kappa B-expressing cells stimulated the induced level of DNA-binding activity, reflecting interaction between endogenous murine and transfected human NF-kappa B proteins. Interestingly, expression of RelA (p65), c-Rel, NF-kappa B1 (p105), NF-kappa B2 (p100), and NF-kappa B2 (p52) subunits increased I kappa B alpha protein levels from 3- to 30-fold, indicating that one mechanism to compensate for the increased expression of NF-kappa B proto-oncogenes was to increase the synthesis and/or stability of the regulatory I kappa B alpha protein. In addition, overexpression of RelA (p65), c-Rel, NF-kappa B2 (p100), and NF-kappa B2 (p52) altered the induction kinetics of IFN-beta mRNA after Sendai virus infection, whereas overexpression of NF-kappa B1 (p105) dramatically decreased IFN-beta mRNA induction. PMID: 8547225 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 360: Mol Cell Biol. 1995 Aug;15(8):4260-71. RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter. Lai JH, Horvath G, Subleski J, Bruder J, Ghosh P, Tan TH. Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA. T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors. PMID: 7623820 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 361: Oncogene. 1995 Jul 6;11(1):97-106. Conserved kappa B element located downstream of the tumor necrosis factor alpha gene: distinct NF-kappa B binding pattern and enhancer activity in LPS activated murine macrophages. Kuprash DV, Udalova IA, Turetskaya RL, Rice NR, Nedospasov SA. Biological Carcinogenesis and Development Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland, 21702-1201, USA. Transcriptional activation of various genes by lipopolysaccharide (LPS) is known to be mediated, at least in part, by the NF-kappa B/Rel family of transcription factors. We have identified a novel kappa B element located immediately downstream of the TNF-alpha gene that is conserved together with its flanking sequences across species lines and can act as an LPS-responsive enhancer for reporter gene constructs driven by the minimal TNF promoter. In extracts from activated murine macrophages and macrophage cell lines this element binds several non-canonical NF-kappa B/Rel complexes, in addition to p50 (NFKB1) homodimer and p50-p65 (NKFB1-RelA) heterodimer. Combination of high-resolution electrophoretic mobility shift assays (EMSA) with monospecific antibodies and u.v.-cross-linking indicates that the prominent slow migrating complex III contain p65 homodimer and c-Rel. The appearance of complex III in EMSA parallels the translocation of p65 and c-Rel into the nucleus and occurs shortly after LPS induction. Transfection experiments with reporter constructs driven by this kappa B element indicate strong inducibility by LPS and p65, moderate inducibility by c-Rel and repression by p50. Functional activity of sandwich TNF-CAT-TNF constructs further suggests that LPS-inducible transcriptional activation of the TNF gene in murine macrophages may be partly mediated by a downstream enhancer. PMID: 7624137 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 362: Mol Cell Biol. 1995 Jul;15(7):3523-30. Overexpression of RelA in transgenic mouse thymocytes: specific increase in levels of the inhibitor protein I kappa B alpha. Perez P, Lira SA, Bravo R. Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA. RelA (p65) is one of the strongest activators of the Rel/NF-kappa B family. As a first step to elucidate the mechanisms that regulate its activity in vivo, we have generated transgenic mice overexpressing RelA in the thymus. Although the levels of RelA were significantly increased in thymocytes of transgenic mice, the overall NF-kappa B-binding activity in unstimulated cells was not augmented compared with that in control thymocytes. This could be explained by the dramatic increase of endogenous I kappa B alpha levels observed in RelA-overexpressing cells in both cytoplasmic and nuclear compartments. The ikba mRNA levels were not augmented by overexpressed RelA, but I kappa B alpha inhibitor was found to be stabilized through association with RelA. Although a fraction of RelA was associated with cytoplasmic p105, no changes in the precursor levels were observed. Upon stimulation of RelA-overexpressing thymocytes with phorbol 12-myristate 13-acetate and lectin (phytohemaglutinin), different kappa B-binding complexes, including RelA homodimers, were partially released from I kappa B alpha. Association of RelA with I kappa B alpha prevented complete degradation of the inhibitor. No effect of phorbol 12-myristate 13-acetate-lectin treatment was detected on RelA associated with p105. Our data indicate that cytoplasmic retention of overexpressed RelA by I kappa B alpha is the major in vivo mechanism controlling the potential excess of NF-kappa B activity in long-term RelA-overexpressing thymocytes. PMID: 7791759 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 363: J Virol. 1995 Jul;69(7):4572-6. Expression of LMP1 in epithelial cells leads to the activation of a select subset of NF-kappa B/Rel family proteins. Paine E, Scheinman RI, Baldwin AS Jr, Raab-Traub N. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295, USA. This study demonstrates that the Epstein-Barr virus protein LMP1 activates a specific subset of NF-kappa B/Rel proteins in the C33 epithelial cell line. Western immunoblot analysis used to analyze the intracellular distribution and abundance of the proteins present in these complexes demonstrated that levels of the p50 and p52 proteins were significantly elevated in the nuclei of LMP1-expressing cells. The data also suggest that LMP1 facilitates the translocation of p50 to the nucleus and may affect the processing of the p100 and p105 precursor proteins or the stability of p52 and p50. PMID: 7769726 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 364: Cell Growth Differ. 1995 Jul;6(7):789-98. Genetic analysis of growth inhibition by GAL4-L kappa B-alpha in Saccharomyces cerevisiae. Morin PJ, Downs JA, Snodgrass AM, Gilmore TD. Department of Biology, Boston University, Massachusetts 02215, USA. I kappa B proteins bind to and regulate Rel/NF- kappa B transcription factors. We showed previously that a fusion protein (GAL4-p40) containing the DNA-binding domain of GAL4 and sequences of chicken l kappa B-alpha (p40) inhibits growth in the yeast Saccharomyces cerevisiae. We now show that p40 must be bound to DNA to inhibit yeast growth, p40 proteins, bound to DNA either as GAL4 or LEXA fusion proteins, inhibit yeast growth. In contrast, p40 proteins that cannot bind to DNA, such as full-length p40, a GAL4-l kappa B fusion protein containing a mutant GAL4 DNA-binding domain, and a fusion protein (GAD-p40) containing the transcriptional activation domain of GAL4 fused to p40, each failed to inhibit cell growth. As with GAL4-VP16, GAL4-p40 needs a functional cellular ADA2 gene to exert its growth-inhibitory effect in S. cerevisiae. Using a high copy suppression strategy, we have isolated three S. cerevisiae genes that restore normal growth to yeast expressing GAL4-p40 or LEXA-p40. We have termed these rescuing genes collectively as SIK genes, for "Suppressors of 1 kappa B." Expression of the SIK genes specifically suppresses the growth-inhibitory activity of GAL4-p40 and LEXA-p40 because SIK gene expression cannot block GAL4-VP16-mediated growth inhibition in S. cerevisiae. SIK1 encodes a novel protein that contains a COOH-terminal repeat that has been found in many microtubule-binding proteins. SIK2 encodes NH2-terminal acetyltransferase, and SIK3 encodes the yeast ribosomal S4 protein. None of the SIK proteins binds directly to p40 sequences in vitro, suggesting that the SIK proteins are likely to act downstream of the direct point of growth inhibition by GAL4-p40. Our results may be useful for devising strategies for identifying vertebrate inhibitors of l kappa B proteins and of other proteins that inhibit growth in S. cerevisiae. PMID: 7547500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 365: J Biol Chem. 1995 Jun 30;270(26):15576-84. Transactivation domain 2 (TA2) of p65 NF-kappa B. Similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. Schmitz ML, dos Santos Silva MA, Baeuerle PA. Institute of Biochemistry, Albert-Ludwigs-University, Freiburg, Germany. The p65 subunit of the inducible transcription factor NF-kappa B contains at least two strong transactivation domains (TADs) within its C terminus. The first domain, TA1, is contained within the last 30 amino acids of p65, whereas TA2 comprises the adjacent 90 amino acids. In this study, squelching experiments revealed that both TADs of p65, as well as the related subunit c-Rel, compete for the same cofactor(s) mediating transactivation. Both TADs of p65 share a common sequence motif, which is evolutionarily conserved and displays a remarkable degree of spatial organization when aligned on an alpha-helical surface. The functional importance of the common sequence motif was confirmed by deletion analysis of TA2. Within the conserved sequence motif, a 7-amino-acid repeat was noted. Idealized heptad repeats fused to the DNA binding domain of Gal4 were transcriptionally active, but only as multimers. Phosphorylation and transcriptional activity of a defined region within the TA2 domain was found to be stimulated by phorbol ester treatment of cells. In contrast, TA1 was constitutively phosphorylated, and its activity did not significantly respond to phorbol ester stimulation. The stimulatory effect of phorbol ester on transcription of the TA2 domain was completely blocked by the protein kinase C inhibitor. These data suggest that protein kinase C has a dual effect on NF-kappa B activity. It not only causes removal of I kappa B-alpha from cytoplasmic NF-kappa B but also augments the transactivation potential of activated nuclear NF-kappa B. PMID: 7797554 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 366: EMBO J. 1995 May 1;14(9):1991-2004. RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A. Neumann M, Grieshammer T, Chuvpilo S, Kneitz B, Lohoff M, Schimpl A, Franza BR Jr, Serfling E. Institute of Pathology, University of Wurzburg, Germany. Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes. PMID: 7744006 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 367: Biochem Biophys Res Commun. 1995 Apr 6;209(1):73-9. Transcriptional inhibition of the inducible nitric oxide synthase gene by competitive binding of NF-kappa B/Rel proteins. Goldring CE, Narayanan R, Lagadec P, Jeannin JF. Laboratoire de l'Ecole Pratique des Hautes Etudes, EPHE, Faculte de Medecine, Dijon, France. The activity of the inducible nitric oxide synthase enzyme (iNOS) is tightly controlled, partly at the transcriptional level. We find NF-kappa B/Rel activation (p50-p50 and p50-p65) in RAW 264.7 macrophages after lipopolysaccharide treatment and binding to both NF-kappa B sites in the mouse iNOS promoter. To delineate the importance of NF-kappa B/Rel in iNOS gene transcription, we used an unusually direct approach to try to improve on the antioxidant-treatment or reporter techniques, namely the depletion of NF-kappa B/Rel activity through the use of a phosphorothioate-modified oligonucleotide containing three copies of the NF-kappa B consensus sequence. The reduction in NF-kappa B/Rel activity (particularly that binding to the downstream of the two sites) was associated with a 50% reduction in NO output and a reduction in the quantity of the iNOS protein expressed. These results point to the probability that physiologically relevant NF-kappa B/Rel activators or repressors other than lipopolysaccharide might crucially affect the macrophage NO response. PMID: 7537042 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 368: Mol Endocrinol. 1995 Apr;9(4):401-12. Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids. Caldenhoven E, Liden J, Wissink S, Van de Stolpe A, Raaijmakers J, Koenderman L, Okret S, Gustafsson JA, Van der Saag PT. Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht. Glucocorticoids are efficient antiinflammatory agents, and their effects include transcriptional repression of several cytokines and adhesion molecules. Whereas glucocorticoids down-regulate the expression of genes relevant during inflammation, nuclear factor (NF)-kappa B/Rel proteins function as important positive regulators of these genes. The expression of intercellular adhesion molecule-1 (ICAM-1), which plays an essential role in recruitment and migration of leukocytes to sites of inflammation, is also down-regulated by glucocorticoids. We found that a functional NF-kappa B site in the ICAM-1 promoter, which can be activated by either 12-O-tetradecanoylphorbol-13-acetate or tumor necrosis factor-alpha (TNF alpha), is also the target for glucocorticoids. In this report we present evidence that the ligand-activated glucocorticoid receptor (GR) is able to repress RelA-mediated activation of the ICAM-1 NF-kappa B site. Conversely, transcriptional activation by GR via a glucocorticoid response element is specifically repressed by RelA, but not by other NF-kappa B/Rel family members. Mutational analysis of GR demonstrates that the DNA binding domain and the ligand binding domain are required for the functional repression of NF-kappa B activation. Despite the importance of the DNA binding domain, we found that the transcriptional repression of NF-kappa B, mediated by GR, is not caused by binding of GR to the ICAM-1 NF-kappa B element, but by a physical interaction between the GR and RelA protein. The repressive effect of GR on NF-kappa B-mediated activation was not shared by other steroid/thyroid receptors. Only the progesterone receptor, which belongs to the same subfamily as GR and which possesses high homology with GR, was able to repress NF-kappa B-mediated transcription. These studies highlight a possible molecular mechanism that can explain the antiinflammatory effects of glucocorticoid treatment during inflammation. PMID: 7659084 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 369: Virology. 1995 Mar 10;207(2):362-8. The v-Rel oncoprotein complexes with new Rel- and RelA-related proteins in transformed cells. Xu X, Gelinas C. Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5638. The v-Rel oncoprotein of the Rev-T retrovirus interacts with a number of cellular proteins in transformed chicken spleen cells including p40/I kappa B alpha, p68c-Rel, hsc70, and the p124 and p115 precursors for the p50 and p52 subunits of NF kappa B. Here we report that v-Rel associates with at least three other cellular proteins of 75-85 kDa in these cells, as well as with a protein related to human RelA. Western blot analysis of v-Rel immune complexes showed cross-reactivity between all of these factors and antibodies raised against Rel sequences, but none appeared to represent isoforms of c-Rel. Synchronization experiments revealed that the expression and/or association of these proteins with v-Rel varied throughout the cell cycle. Combined with the previously described interaction of v-Rel with known members of the Rel family, these studies strengthen the hypothesis that the interaction of v-Rel with multiple Rel-related proteins may be important for the transformation of lymphoid cells. PMID: 7886940 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 370: Mol Cell Biol. 1995 Mar;15(3):1294-301. In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B. Alkalay I, Yaron A, Hatzubai A, Jung S, Avraham A, Gerlitz O, Pashut-Lavon I, Ben-Neriah Y. Lautenberg Center for General and Tumor Immunology, Hebrew University, Hadassah Medical School, Jerusalem, Israel. NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation. PMID: 7862123 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 371: J Biol Chem. 1995 Feb 24;270(8):3849-57. Regulation of the tissue factor promoter in endothelial cells. Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors. Moll T, Czyz M, Holzmuller H, Hofer-Warbinek R, Wagner E, Winkler H, Bach FH, Hofer E. Department of Transplantation Immunology, Vienna International Research Cooperation Center, Vienna, Austria. Tissue factor is up-regulated on endothelial cells and monocytes in response to cytokines and endotoxin and is the main trigger of the extrinsic pathway of the coagulation cascade. We have isolated the porcine tissue factor gene and studied the regulation of the promoter, which has not been investigated previously in endothelial cells. Comparison of the promoter sequences with the respective human and murine genes reveals short stretches of homology, which encompass potential binding sites for AP-1, NF kappa B, and Sp1 transcription factors. Using DNase I footprinting, we detect binding of nuclear factors to these promoter elements. Transfection experiments demonstrate that a 300-base pair fragment containing the conserved elements can mediate induced transcription and that the NF kappa B-like element is essential. In accordance, electrophoretic mobility shift assays show a strong increase in the binding of factors to the NF kappa B-like site following induction. We further provide evidence that RelA (p65), c-Rel, and possibly novel polypeptides bind to the tissue factor NF kappa B element. In addition, we show constitutive binding of members of the Fos/Jun and Sp1 families to the AP-1 and Sp1 sites, respectively. We propose a concerted action of AP-1-, NF kappa B-, and Sp1-like factors in transcription from the tissue factor promoter in endothelial cells. PMID: 7876129 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 372: J Biol Chem. 1995 Feb 10;270(6):2703-7. Tumor necrosis factor-alpha-dependent activation of a RelA homodimer in astrocytes. Increased phosphorylation of RelA and MAD-3 precede activation of RelA. Diehl JA, Tong W, Sun G, Hannink M. Biochemistry Department, University of Missouri, Columbia 65212. Rel proteins are important intracellular mediators of cytokine-induced signal transduction. To understand how cytokines affect different cell populations in the brain, we have characterized Rel activation in astrocytes. A RelA homodimer is uniquely activated in cytokine-stimulated astrocytes. Cytokine-dependent phosphorylation of the RelA inhibitor MAD-3 occurred on discrete peptides prior to its dissociation from RelA. A transient hyperphosphorylation of RelA was also induced. Antioxidant treatment inhibited both RelA activation and phosphorylation of the RelA.MAD-3 complex. These results demonstrate that cytokine-dependent activation of the RelA homodimer involves phosphorylation of both RelA and its associated inhibitor. The sole activation of a RelA homodimer suggests that cytokines will activate a unique set of Rel-regulated genes in astrocytes. PMID: 7852340 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 373: Mol Cell Biol. 1995 Feb;15(2):943-53. Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599. Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription. PMID: 7823959 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 374: Mol Cell Biol. 1995 Feb;15(2):872-82. The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers. Ernst MK, Dunn LL, Rice NR. Laboratory of Molecular Virology and Carcinogenesis, National Cancer Institute Frederick Cancer Research and Development Center, Maryland 21702-1201. In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer. PMID: 7823953 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 375: J Leukoc Biol. 1995 Jan;57(1):174-9. Macrophages derived from C3H/HeJ (Lpsd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B. Ding A, Hwang S, Lander HM, Xie QW. Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York. The effects of bacterial lipopolysaccharide (LPS) on macrophage gene expression are mediated in part by its ability to induce activation of transcription factor NF-kappa B. We compared the ability of LPS-treated macrophages from Lpsn (LPS-responsive) C3H/HeN and Lpsd (LPS-hyporesponsive) C3H/HeJ mice to mobilize NF-kappa B by electrophoretic mobility shift assays with oligonucleotide probes containing a unique NF-kappa B sequence from the promoter of inducible nitric oxide synthase (iNOS). In response to ng/ml concentrations of LPS, this probe bound proteins that appeared rapidly in the nuclei of thioglycollate-elicited macrophages and bone marrow-derived macrophage cell lines from both Lpsn and Lpsd mice. Only in macrophages from Lpsn mice, however, was LPS able to induce iNOS or tumor necrosis factor alpha. NF-kappa B-containing DNA-protein complexes from Lpsd macrophages were formed in lesser amounts than from Lpsn macrophages but shared the same composition, insofar as they displayed the same electrophoretic mobilities and content of heterodimers of p50/RelA (p65) and p50/c-rel. Two conclusions emerge from these findings: (1) NF-kappa B activity alone is not sufficient for induction of certain LPS-responsive genes and (2) An LPS-response pathway involving activation of NF-kappa B is preserved in Lpsd mice. The inability of cells from Lpsd mice to induce gene expression in response to LPS thus cannot be attributed to inability to activate NF-kappa B. PMID: 7829969 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 376: Cancer Biochem Biophys. 1995 Jan;14(4):265-72. Activation of NF-kappa B in murine macrophages by taxol. Hwang S, Ding A. Beatrice and Samuel A. Seaver Laboratory, Cornell University Medical College, New York, New York 10021, USA. Taxol, a plant-derived antimitotic, was recently found to mimic several of the effects of endotoxic bacterial lipopolysaccharide on murine macrophages. However, the mechanisms underlying the cell cycle-independent actions of taxol remain unclear. Here, we report that taxol rapidly activated nuclear factor kappa B (NF-kappa B) in mouse peritoneal macrophages. The intranuclear transcription factor complexes contained two NF-kappa B heterodimers, p50/RelA and p50/c-rel. Taxol-induced nuclear translocation of NF-kappa B was inhibited by pyrrolidine dithiocarbamate, an antioxidant, but not by cycloheximide, a protein synthesis inhibitor. The ability of taxol to activate NF-kappa B may help account for its induction of immunoregulatory and cytotoxic cytokines, which in turn may contribute to its antitumor effects. PMID: 7767900 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 377: J Biol Chem. 1994 Dec 23;269(51):32162-7. Structural and functional analysis of NF-kappa B. Determinants of DNA binding specificity and protein interaction. Schmid RM, Liptay S, Betts JC, Nabel GJ. Howard Hughes Medical Institute, University of Michigan Medical Center, Department of Internal Medicine, Ann Arbor 48109-0650. The NF-kappa B transcription factors display a high degree of sequence conservation in a domain initially described in the rel oncogene. Two family members, NF-kappa B1 and NF-kappa B2, have distinct DNA binding properties and functionally distinct effects on different enhancers. NF-kappa B1, for example, binds to the kappa B site from the human immunodeficiency virus (HIV) with approximately 15-fold higher affinity than NF-kappa B2. In this study, we have defined regions within the Rel domain which determine DNA binding specificity and interaction with other proteins. We find that the COOH-terminal putative Rel dimerization domain of NF-kappa B1 is required for preferential binding to the HIV kappa B site. In contrast, preferential stimulation of the HIV enhancer by NF-kappa B2 with RelA(p65) is determined by both the NH2- and COOH-terminal Rel domains of NF-kappa B2. These two regions of NF-kappa B2 also mediate preferential synergy with Bcl3. These data suggest that a specific subdomain of the Rel conserved region has evolved to control the fine specificity of DNA binding, and two distinct subregions within the Rel domain determine the specificity of interaction with other transcription factors. These specific Rel-conserved domains therefore determine the specificity of NF-kappa B interactions and contribute to selective gene activation. PMID: 7798213 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 378: J Biol Chem. 1994 Dec 2;269(48):30429-35. Rapid activation of post-hepatectomy factor/nuclear factor kappa B in hepatocytes, a primary response in the regenerating liver. Cressman DE, Greenbaum LE, Haber BA, Taub R. Department of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6145. The liver represents one of the few organs in the intact animal that has the capacity to regenerate following injury or partial hepatectomy. One of the earliest responses that has been detected in the remnant liver is the activation of post-hepatectomy factor(s) (PHF), a kappa B site DNA binding activity. We reasoned that understanding the molecular nature of PHF might provide insight into what triggers liver regeneration. We found that PHF is rapidly activated and turned over in the regenerating liver, demonstrating peak activity at 30 min post-hepatectomy and virtual disappearance by 1 h. As determined by supershift, cross-linking, and cross-linking/immunoprecipitation analyses, PHF contains intact p50/p65nuclear factor kappa B (NF-kappa B) subunits. To explore the basis for activation of PHF/NF-kappa B in the regenerating liver, we determined the level of individual Rel family subunits in the nuclei of normal and regenerating liver cells. We found evidence for nuclear translocation of p65/RelA, but other Rel family proteins including p50/NF-kappa B1 and p52/NF-kappa B2 are present at a low level in the nuclei of cells at a constitutive level pre- and post-hepatectomy and appear not to form DNA binding homodimers. The level of I kappa B-alpha falls slightly then increases at 3 h post-hepatectomy in concert with the induction of its mRNA. As demonstrated by the induction of I kappa B-alpha mRNA in hepatocytes in situ and identification of PHF/NF-kappa B in cultured hepatocytes, PHF/NF-kappa B is localized primarily in hepatocytes in the regenerating liver. This represents one of the few examples of NF-kappa B activation in the intact animal in a non-hematopoietic cell type. The activation of PHF/NF-kappa B suggests a mechanism by which hepatocytes regulate their mitogenic program during liver regeneration. PMID: 7982957 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 379: Mol Cell Biol. 1994 Dec;14(12):7933-42. Effect of CD28 signal transduction on c-Rel in human peripheral blood T cells. Bryan RG, Li Y, Lai JH, Van M, Rice NR, Rich RR, Tan TH. Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030. Optimal T-cell activation requires both an antigen-specific signal delivered through the T-cell receptor and a costimulatory signal which can be delivered through the CD28 molecule. CD28 costimulation induces the expression of multiple lymphokines, including interleukin 2 (IL-2). Because the c-Rel transcription factor bound to and activated the CD28 response element within the IL-2 promoter, we focused our study on the mechanism of CD28-mediated regulation of c-Rel in human peripheral blood T cells. We showed that CD28 costimulation accelerated the kinetics of nuclear translocation of c-Rel (and its phosphorylated form), p50 (NFKB1), and p65 (RelA). The enhanced nuclear translocation of c-Rel correlated with the stimulation of Il-2 production and T-cell proliferation by several distinct anti-CD28 monoclonal antibodies. This is explained at least in part by the long-term downregulation of I kappa B alpha following CD28 signalling as opposed to phorbol myristate acetate alone. Furthermore, we showed that the c-Rel-containing CD28-responsive complex is enhanced by, but not specific to, CD28 costimulation. Our results indicate that c-Rel is one of the transcription factors targeted by CD28 signalling. PMID: 7969133 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 380: J Immunol. 1994 Nov 15;153(10):4713-20. Murine inhibitory protein-kappa B alpha negatively regulates kappa B-dependent transcription in lipopolysaccharide-stimulated RAW 264.7 macrophages. Tebo JM, Chaoqun W, Ohmori Y, Hamilton TA. Department of Immunology, Cleveland Clinic Foundation, Ohio 44195. The potential role of the inhibitory protein (I)-kappa B alpha gene in control of LPS-dependent transcription has been investigated in the murine macrophage cell line RAW 264.7. LPS-induced transcription in macrophages is believed to involve activation of members of the Rel homology family of transcription factors, and may be negatively regulated by cytoplasmic inhibitor proteins collectively termed I-kappa Bs. To evaluate the role of I-kappa Bs in LPS-stimulated macrophages, murine I-kappa B alpha (ml-kappa B alpha) has been expressed as a glutathione-S-transferase (GST) fusion protein and examined for its ability to control kappa B binding activities in nuclear extracts from LPS-treated RAW 264.7 macrophages. ml-kappa B alpha-GST inhibited LPS-induced kappa B binding activity from RAW 264.7 cells in a phosphorylation-dependent fashion, but did not affect IFN-alpha-induced IFN stimulus response element binding. Recombinant I-kappa B alpha inhibited kappa B motif binding by nuclear factor-kappa B1, RelA, and c-Rel as indicated by studies using UV radiation-induced covalent cross-linking to a bromodeoxyuridine-substituted kappa B oligonucleotide. Transfection of macrophages with an expression vector encoding ml-kappa B alpha inhibited LPS-stimulated transcription driven by a 243-bp promoter sequence obtained from the 5' flanking region of the murine IP-10 gene. This promoter sequence contains two kappa B motifs that have been shown to be critical to LPS-dependent reporter gene transcription. The kappa B sites seem to be the specific target of I-kappa B alpha function, as reporter gene transcription driven by these motifs in the context of a heterologous thymidine-kinase promoter (TK) also was inhibited by co-transfection with ml-kappa B alpha. These observations indicate that ml-kappa B alpha is capable of controlling kappa B-dependent transcription in LPS-stimulated murine macrophages. PMID: 7963540 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 381: J Immunol. 1994 Nov 15;153(10):4357-66. Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B. Berberich I, Shu GL, Clark EA. Department of Microbiology, University of Washington, Seattle 98195. The B cell-associated surface molecule CD40 functions to regulate B cell responses. Cross-linking CD40 on B cells can lead to homotypic cell adhesion, IL-6 production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by CD40 cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF-kappa B-like transcription factors are activated after cross-linking CD40 on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (RelA), c-Rel, and most likely other components. By using transient transfection assays, we found that cross-linking CD40 supports NF-kappa B-dependent gene expression. Our results define the NF-kappa B system as an intermediate event in CD40 signaling and suggest that the CD40 pathway can influence the expression of B cell-associated genes with NF-kappa B consensus sites. PMID: 7525701 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 382: Oncogene. 1994 Nov;9(11):3289-97. Constitutive and inducible Rel/NF-kappa B activities in mouse thymus and spleen. Weih F, Carrasco D, Bravo R. Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000. We have studied the expression of members of the rel family of transcription factors and ikba in mouse thymus and spleen by in situ hybridization. Our results show that the rel genes have different temporal and spatial patterns of expression suggesting distinct roles in these lymphoid tissues. The Rel/NF-kappa B proteins and I kappa B alpha in thymus and spleen were also analysed by Western blotting and electrophoretic mobility shift assays. Although RelB protein is present at significantly lower levels in thymus and spleen extracts when compared to RelA, in both tissues the predominant kappa B-binding activity consists of p50/RelB and p52/RelB heterodimers and only very little binding of RelA-containing complexes to kappa B sites was detected. Significant binding of c-Rel complexes was only found in spleen extracts. Treatment of thymus and spleen extracts with deoxycholate (DOC), however, results in a strong increase in binding to kappa B sites of both RelA and c-Rel complexes. In contrast, binding of RelB complexes is not induced after DOC treatment. Our results suggest a differential role of Rel/NF-kappa B complexes in mouse thymus and spleen with RelB heterodimers representing the constitutive kappa B-binding activity, whereas RelA and c-Rel complexes most likely are involved in inducible kappa B-binding and gene activation. PMID: 7936653 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 383: Oncogene. 1994 Nov;9(11):3099-105. Tax protein of HTLV-1 interacts with the Rel homology domain of NF-kappa B p65 and c-Rel proteins bound to the NF-kappa B binding site and activates transcription. Suzuki T, Hirai H, Yoshida M. Department of Cellular and Molecular Biology, University of Tokyo, Japan. Tax protein of human T cell leukemia virus type 1 (HTLV-1) enhances transcription of several cellular genes through activation of a specific enhancer, the NF-kappa B binding site. We found previously that Tax binds to NF-kappa B p50, which is a member of the Rel/NF-kappa B family proteins, and associates with the DNA sequence of the NF-kappa B binding site. In the present study, we tested other NF-kappa B family proteins and found that NF-kappa B p65 and c-Rel proteins also bind to Tax and that their complexes with Tax bind to the DNA sequence of the NF-kappa B binding site. The Tax binding site on NF-kappa B p50 is the Rel homology domain, which is conserved in the Rel/NF-kappa B family proteins. The formations of these complexes by Tax mutants were well correlated with their transactivating capacities. In F9 embryonic carcinoma cells, Tax enhanced transcription synergistically with NF-kappa B p65 or c-Rel. Thus Tax interacts with the Rel homology domain of Rel/NF-kappa B family proteins which bind to the NF-kappa B binding site and activates transcription. PMID: 7936632 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 384: Mol Cell Biol. 1994 Nov;14(11):7377-84. Human T-cell leukemia virus type I Tax activation of NF-kappa B/Rel involves phosphorylation and degradation of I kappa B alpha and RelA (p65)-mediated induction of the c-rel gene. Sun SC, Elwood J, Beraud C, Greene WC. Gladstone Institute of Virology and Immunology, University of California, San Francisco 94141-9100, USA. The tax gene product of human T-cell leukemia virus type I (HTLV-I) is a potent transcriptional activator that both stimulates viral gene expression and activates an array of cellular genes involved in T-cell growth. Tax acts indirectly by inducing or modifying the action of various host transcription factors, including members of the NF-kappa B/Rel family of enhancer-binding proteins. In resting T cells, many of these NF-kappa B/Rel factors are sequestered in the cytoplasm by various ankyrin-rich inhibitory proteins, including I kappa B alpha. HTLV-I Tax expression leads to the constitutive nuclear expression of biologically active NF-kappa B and c-Rel complexes; however, the biochemical mechanism(s) underlying this response remains poorly understood. In this study, we demonstrate that Tax-stimulated nuclear expression of NF-kappa B in both HTLV-I-infected and Tax-transfected human T cells is associated with the phosphorylation and rapid proteolytic degradation of I kappa B alpha. In contrast to prior in vitro studies, at least a fraction of the phosphorylated form of I kappa B alpha remains physically associated with the NF-kappa B complex in vivo but is subject to rapid degradation, thereby promoting the nuclear translocation of the active NF-kappa B complex. We further demonstrate that Tax induction of nuclear c-Rel expression is activated by the RelA (p65) subunit of NF-kappa B, which activates transcription of the c-rel gene through an intrinsic kappa B enhancer element. In normal cells, the subsequent accumulation of nuclear c-Rel acts to inhibit its own continued production, indicating the presence of an autoregulatory loop. However, the pathologic action HTLV-I Tax leads to the deregulated and sustained nuclear expression of both NF-kappa B and c-Rel, a response that may contribute to HTLV-I-induced T-cell transformation. PMID: 7935451 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 385: J Virol. 1994 Nov;68(11):7131-8. Interaction of the v-Rel oncoprotein with cellular transcription factor Sp1. Sif S, Gilmore TD. Department of Biology, Boston University, Massachusetts 02215. We previously showed that v-Rel, the oncoprotein of the avian retrovirus Rev-T, can increase expression from promoters containing binding sites for the cellular transcription factor Sp1 in chicken embryo fibroblasts (S. Sif, A.J. Capobianco, and T.D. Gilmore, Oncogene 8:2501-2509, 1993). In those experiments, v-Rel appeared to increase the transactivating function of Sp1; that is, v-Rel stimulated transactivation by a GAL4-Sp1 protein that lacked the Sp1 DNA-binding domain. We have now shown that in vitro-synthesized v-Rel and GAL4-Sp1 form a complex that can be immunoprecipitated with either anti-Sp1 or anti-v-Rel antiserum. We have also shown that a glutathione S-transferase (GST)-Sp1 fusion protein can specifically interact with in vitro-translated v-Rel and with in vivo-synthesized v-Rel from transformed chicken spleen cells. In addition, we have found that the abilities of wild-type and two mutant forms of v-Rel to increase transactivation by Sp1 in vivo correlate with their abilities to interact with Sp1 in vitro. The sequences important for the interaction of v-Rel with Sp1 in vitro have been mapped to the first 147 amino acids of v-Rel. Other Rel proteins, such as c-Rel, RelA, p52, and p50, were also able to form a complex with Sp1 in vitro. These results suggest that v-Rel increases expression from Sp1 site-containing promoters by functionally interacting with Sp1 and that cellular Rel proteins and Sp1 are likely to interact to influence transcription from natural promoters. PMID: 7933095 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 386: J Immunol. 1994 Oct 15;153(8):3584-93. LPS-induced expression of the human IL-1 receptor antagonist gene is controlled by multiple interacting promoter elements. Smith MF Jr, Eidlen D, Arend WP, Gutierrez-Hartmann A. Department of Medicine, University of Colorado Health Sciences Center, Denver 80262. Expression of the IL-1 receptor antagonist (IL-1ra) can be induced by treatment of monocytes and macrophages with LPS. We have previously demonstrated that the most proximal 294 bp of the human IL-1ra promoter are sufficient for full basal activity and LPS responsiveness. In the present study, we demonstrate the presence of one inhibitory and three positive-acting LPS response elements (LRE) within this proximal 294-bp IL-1ra promoter fragment. By both 5'-deletional analysis and heterologous promoter studies, an element between -294 and -250 was found to mask the LPS response. By 5'-deletional analysis and heterologous promoter experiments, two positive-acting LRE were identified between -250 and -200 (LRE3) and -200 and -148 (LRE2) which exhibited cooperativity in that neither element alone was active. Furthermore, LRE2 also cooperated with a more proximal site between -148 and -31 (LRE1), which also was not active alone. LRE1 was identified as an NF-kappa B-binding site. Site-directed mutagenesis of this site, located between -93 and -84, resulted in a > 50% decrease in the LPS responsiveness of the 294-bp promoter. By electrophoretic mobility shift assays, with or without specific antisera to members of the rel/NF-kappa B family, the complex binding to LRE1 was shown to contain primarily NF-kappa B1/p50 and lesser amounts of RelA/p65. These results indicate that the net activation of the human IL-1ra promoter in response to LPS involves the functional interaction of at least four cis-acting DNA elements within the proximal 294 bp. PMID: 7930581 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 387: Cancer Res. 1994 Oct 15;54(20):5424-9. T cells from renal cell carcinoma patients exhibit an abnormal pattern of kappa B-specific DNA-binding activity: a preliminary report. Li X, Liu J, Park JK, Hamilton TA, Rayman P, Klein E, Edinger M, Tubbs R, Bukowski R, Finke J. Department of Immunology, Cleveland Clinic Foundation, Ohio 44195. Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (p50) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that RelA, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any RelA in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients may impair their capacity to respond normally to various stimuli. PMID: 7923175 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 388: J Biol Chem. 1994 Oct 14;269(41):25613-20. Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation. Schmitz ML, dos Santos Silva MA, Altmann H, Czisch M, Holak TA, Baeuerle PA. Institute for Biochemistry, Albert Ludwigs University, Freiburg, Federal Republic of Germany. The p65 subunit of the NF-kappa B transcription factor contains in its C-terminal 120 amino acids at least two transcription activation domains. One domain (TA1) is contained within only the 30 C-terminal amino acids. Structural studies employing CD and NMR spectroscopy revealed that the TA1 domain is unstructured. NMR analysis of a protein corresponding to the C-terminal 123 amino acids also showed a random coil conformation. However, a portion of TA1 was found to adopt an alpha-helical conformation in the presence of hydrophobic solvents. Transcriptional analysis of point mutants revealed the functional importance of two evolutionary conserved sequence repeats, which are located in the conditionally alpha-helical region of TA1. These repeats acted synergistically in transcription activation. The inhibitory effect of some mutants indicated secondary structure constraints on TA1 in intact cells. Inverting the sequence of two acidic activation domains significantly reduced their transactivating potential, suggesting that amino acid composition is not solely essential for activity; a defined primary structure is necessary as well. Acidic sequence motifs related in primary structure and squelching activity to those of TA1 are present in the activation domains of VP16, c-Rel, and several other transcription factors. We propose a model suggesting that primarily unstructured acidic activation domains can adopt a secondary structure upon contacting their target molecules by an "induced fit" mechanism. PMID: 7929265 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 389: Mol Cell Biol. 1994 Oct;14(10):6635-46. Identification of a C/EBP-Rel complex in avian lymphoid cells. Diehl JA, Hannink M. Biochemistry Department, University of Missouri, Columbia 65212. Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography. The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site. The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members suggests a novel mechanism for regulation of gene expression by Rel family proteins. PMID: 7935382 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 390: Mol Cell Biol. 1994 Oct;14(10):6443-51. Kinetic analysis of human T-cell leukemia virus type I Tax-mediated activation of NF-kappa B. Kanno T, Brown K, Franzoso G, Siebenlist U. Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892. The human T-cell leukemia virus type I (HTLV-I) Tax protein induces the expression of cellular genes, at least in part, by activating the endogenous NF-kappa B transcription factors. Induced expression of cellular genes is thought to be important for transformation of T cells to continued growth, a prelude to the establishment of adult T-cell leukemia. However, neither underlying mechanisms nor kinetics of the Tax-mediated activation of NF-kappa B are understood. We have analyzed a permanently transfected Jurkat T-cell line in which the expression of Tax is entirely dependent on addition of heavy metals. The initial NF-kappa B binding activity seen after induction of Tax is due almost exclusively to p50/p65 heterodimers. At later times, NF-kappa B complexes containing c-Rel and/or p52 accumulate. The early activation of p50/p65 complexes is a posttranslational event, since neither mRNA nor protein levels of NF-kappa B subunits had increased at that time. We demonstrate for the first time a Tax-induced proteolytic degradation of the NF-kappa B inhibitor, I kappa B-alpha, which may trigger the initial nuclear translocation of NF-kappa B. As nuclear NF-kappa B rapidly and potently stimulates resynthesis of I kappa B-alpha, the steady-state level of I kappa B-alpha does not significantly change. Thus, the dramatic Tax-induced increase in the I kappa B-alpha turnover may continually weaken inhibition and activate NF-kappa B. Additional, distinct actions of Tax may contribute further to the high levels of NF-kappa B activity seen. PMID: 7935369 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 391: J Biol Chem. 1994 Sep 2;269(35):22230-7. Differential DNA sequence specificity and regulation of HIV-1 enhancer activity by cRel-RelA transcription factor. Hansen SK, Guerrini L, Blasi F. Department of Genetics and Microbiol Biology, University of Milano, Italy. The cRel-RelA and NF-kappa B (p50-RelA) transcription factors bind to a kappa B-like sequence termed Rel-related proteins binding element localized in the regulatory region of the human urokinase plasminogen activator (uPA) gene. This sequence is highly conserved in murine and porcine uPA genes where it retained the ability to associate with cRel-RelA. On the other hand, NF-kappa B binding was obtained with the human and porcine elements only. Methylation interference analysis showed that NF-kappa B and cRel-RelA had identical interference patterns. Mutational analysis showed that DNA binding was highly sensitive to mutations within the decameric Rel-related proteins binding element core site. However, alterations of nucleotides flanking the decameric IgK-kappa B motif, which preferentially associated with NF-kappa B, resulted in high affinity cRel-RelA binding both in vitro and in vivo. These data demonstrate that NF-kappa B and cRel-RelA have overlapping but distinct DNA sequence specificities. Bandshift analysis with HeLa and Jurkat cell extracts or with in vitro translated proteins revealed that the SV40-, HIV-1-, and interleukin-2 receptor alpha subunit kappa B elements efficiently associated with cRel-RelA, suggesting that this heterodimer may be involved in the regulation of several genes. Cotransfection studies of HIV-1 long terminal repeat-chloramphenicol acetyltransferase reporter DNA with RelA, cRel, and p50 expression vectors were performed in COS7 and U293 cells to analyze the ability of cRel-RelA to regulate HIV-1 enhancer activity. In vivo formation of the cRel-RelA complex resulted in specific stimulation of the viral enhancer at a level comparable with that obtained with NF-kappa B. These data suggest that activation of cellular cRel-RelA may play a critical role in the regulation of HIV-1 enhancer activity. PMID: 8071349 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 392: EMBO J. 1994 Sep 1;13(17):4060-9. Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells. Lernbecher T, Kistler B, Wirth T. Zentrum fur Molekulare Biologie Heidelberg, Germany. The kappa B-motif is an important regulatory element both for constitutive lymphoid-specific as well as ubiquitous inducible transcriptional activity. We have shown previously that different members of the NF-kappa B/Rel family of transcription factors are responsible for these distinct functions. Whereas the p65/RelA protein is involved in inducible kappa B-dependent transcription, RelB is associated with constitutive activity in lymphoid cells. Here we have addressed the question of how RelB is constitutively activated in lymphoid cells. We demonstrate that this is achieved by two different mechanisms. Expression of relB as determined by measurement of stable RNA and protein levels is significantly enhanced in lymphoid organs compared with non-lymphoid organs. However, these observed differences in absolute amounts of RNA and protein would not suffice to explain the dramatic differences that are apparent at the level of active DNA binding complexes in extracts from the respective organs. We have therefore analyzed the interaction of RelB complexes with the I kappa B-alpha inhibitor protein. Our results show that RelB-containing complexes present in lymphoid extracts are much less susceptible to inhibition by I kappa B-alpha than RelA- or RelB-containing complexes from non-lymphoid cells. This difference might be due to post-translational modifications of the RelB protein or interaction with a lymphoid-specific cofactor for RelB. PMID: 8076601 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 393: J Biol Chem. 1994 Aug 19;269(33):20823-5. A set of inducible genes expressed by activated human monocytic and endothelial cells contain kappa B-like sites that specifically bind c-Rel-p65 heterodimers. Parry GC, Mackman N. Department of Immunology, Scripps Research Institute, La Jolla, California 92037. NF-kappa B/Rel proteins regulate the inducible expression of many genes in activated monocytes and endothelial cells that contain decameric kappa B and kappa B-like binding sites. In this study, we examined the binding of c-Rel-p65 heterodimers to non-consensus kappa B-like sites from several genes that do not bind prototypic NF-kappa B(p50-p65). c-Rel-p65 heterodimers from both monocytic and endothelial cells bound to the kappa B-like sites in the interleukin-8, granulocyte/macrophage colony-stimulating factor, intercellular adhesion molecule-1, and tissue factor genes but not to a closely related sequence in the granulocyte colony-stimulating factor gene. In contrast, kappa B sites in the endothelial-leukocyte adhesion molecule-1 and Ig kappa genes that match the kappa B consensus, 5'-GGGRNNYYCC-3' (where R indicates A or G, Y indicates C or T, and N indicates any base), bound NF-kappa B(p50-p65). Comparison of the kappa B-like sites indicated that c-Rel-p65 heterodimers bound to a consensus sequence, 5'-HGGARNYYCC-3' (where R indicates A or G, Y indicates C or T, H indicates A, C, or T, and N indicates any base), which differs at position 1 from the kappa B consensus established for binding NF-kappa B(p50-p65) and other members of the NF-kappa B/Rel family. The selective binding of c-Rel-p65 heterodimers to kappa B-like sites in this set of genes may play a central role in regulating inducible gene expression in monocytes and endothelial cells. PMID: 8063696 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 394: Cell Immunol. 1994 Aug;157(1):158-69. Long-term inositol phosphate release, but not tyrosine kinase activity, correlates with IL-2 secretion and NF-AT induction in anti-CD3-activated peripheral human T lymphocytes. Bryan RG, Li Y, Totten RK, Van M, Tan TH, Rich RR. Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030. The cascade of events within the first few minutes of T cell stimulation has been well characterized. Although many second messengers have been shown to be necessary and sufficient for T cell activation in a number of model systems, the rate-limiting step in peripheral T cells has not been demonstrated. To model effective versus ineffective CD3-mediated stimulation in peripheral T cells, we used two anti-CD3 mAbs that differ in their ability to stimulate purified T cells: OKT3, which causes early second messenger generation but is unable to activate T cells without a second signal, and 64.1, which stimulates T cell proliferation on its own. We found that tyrosine kinase activity was similar for both mAbs over a period of hours. However, the inositol phosphate response was stronger for 64.1 than for OKT3. To tie these events to gene activation, we measured NF-kappa B and NF-AT activity in the nucleus after anti-CD3 stimulation. Both stimuli induced the appearance of the NF-kappa B components (c-Rel, p65 (RelA), and p50 (NF-kappa B1)) and NF-kappa B DNA binding activity in the nucleus. However, only 64.1 induced NF-AT in the nucleus, correlating with its ability to activate T cells. Thus, NF-AT induction and IL-2 secretion were correlated with the levels of inositol phosphate release but not with gross levels of tyrosine kinase activity induced late following the response. On the other hand, NF-kappa B induction and IL-2 receptor expression occurred even with the smaller second messenger response generated by OKT3. PMID: 8039243 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 395: Mol Cell Biol. 1994 Aug;14(8):5349-59. Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation. Liou HC, Sha WC, Scott ML, Baltimore D. Rockfeller University, New York, New York 10021. The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs. PMID: 8035813 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 396: J Biol Chem. 1994 Jul 1;269(26):17684-90. Kappa B binding activity in a murine macrophage-like cell line. Sequence-specific differences in kappa B binding and transcriptional activation functions. Ohmori Y, Tebo J, Nedospasov S, Hamilton TA. Department of Immunology, Cleveland Clinic Foundation, Ohio 44195. The role of two distinct kappa B sequence motifs found in the promotor of the murine IP-10 gene was studied in the transcriptional response of macrophages to lipopolysaccharides (LPS). When the murine macrophage cell line RAW 264.7 was stimulated with LPS, at least three different kappa B sequence-specific complex-forming activities were observed in nuclear extracts as assayed by electrophoretic mobility shift assay (EMSA). These three complexes were distinguished from one another in terms of time of appearance following stimulation and selectivity for one of the two different kappa B sequence motifs. The participation of individual members of the Rel homology family of kappa B sequence binding factors was assessed by use of specific antibodies in combination with either EMSA or UV-cross-linking to radiolabeled, BrdUrd-substituted oligonucleotide probes. The C1 complex contained predominantly NF kappa B1 (p50). The C2 complex contained NF kappa B1, RelA (p65), and perhaps other factors. The C3 complex contained predominantly c-Rel. Both kappa B sequences were able to mediate reporter gene transcription in LPS-stimulated macrophages, but the sites behaved differentially in cells co-transfected with expression vectors encoding different members of the Rel homology family. The results indicate that LPS activates several different forms of kappa B binding activity in murine macrophages which are composed of at least three different members of the Rel homology family. These binding activities exhibit differential recognition of and functional activation through the two distinct kappa B sequence motifs. PMID: 8021280 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 397: J Biol Chem. 1994 Jun 10;269(23):16236-41. Effects of guanosine 3',5'-bisdiphosphate (ppGpp) on rate of transcription elongation in isoleucine-starved Escherichia coli. Vogel U, Jensen KF. Department of Biological Chemistry, University of Copenhagen, Denmark. We measured the transcription elongation rate on two mRNA genes, i.e. infB and lacZ, and on a part of the rrnB gene under conditions when wild type (rel+) Escherichia coli and relaxed (relA) mutants were exposed to isoleucine starvation. The RNA chain growth rates were calculated from the time lag between induction of transcription and the appearance of specific hybridization to probes complementary to the 3' ends of the genes, i.e. from the transcription time. The rate of mRNA chain elongation responded differently in rel+ and relA strains exposed to isoleucine starvation as it decreased (approximately 50%) in rel+ strains that accumulated high concentrations of guanosine 3',5'-bisdiphosphate (ppGpp) and increased (approximately 15%) the relA mutant whose ppGpp pool decayed during starvation. These results show that ppGpp inhibits mRNA chain elongation in vivo. However, stable RNA chain elongation appeared unaffected by ppGpp pool size and was twice as fast as mRNA chain elongation in exponentially growing cells. PMID: 8206927 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 398: Proc Natl Acad Sci U S A. 1994 May 24;91(11):5056-60. Qualitative changes in the subunit composition of kappa B-binding complexes during murine B-cell differentiation. Miyamoto S, Schmitt MJ, Verma IM. Molecular Biology and Virology Laboratory, Salk Institute, San Diego, CA 92186-5800. We report here that the major kappa B-binding complex in murine mature B cells is composed of a p50-Rel heterodimer, whereas the major inducible form in pre-B cells is a p50-p65 heterodimer. Treatment of a pre-B-cell line with lipopolysaccharide changes the subunit composition of kappa B-binding complexes from p50-p65 to p50-Rel. This change is preceded by the enhanced Rel expression and correlates with the expression of the gene for the immunoglobin kappa light chain. The heterodimeric p50-Rel complex binds to the intronic enhancer-kappa B site in the immunoglobulin kappa light chain gene at least 20-fold more stably than does the p50-p65 dimer. These data support a model in which augmentation of Rel expression during the differentiation of pre-B cells to mature B cells leads to an exchange of kappa B-binding subunits resulting in the transcriptional activation of the gene for the immunoglobulin kappa light chain. PMID: 8197184 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 399: Oncogene. 1994 May;9(5):1487-92. Activation of multiple NF-kappa B/Rel DNA-binding complexes by tumor necrosis factor. Beg AA, Baldwin AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599. NF-kappa B is an inducible transcription factor that regulates the expression of numerous genes involved in immune and inflammation responses and in cellular growth control. Typically, NF-kappa B is localized in the cytoplasm complexed with members of the I kappa B family. The most well characterized form of NF-kappa B is comprised of a heterodimer of a 50 kD (p50/NFKB1) and a 65 kD (p65/RelA) protein. This heterodimeric protein was thought to be primarily responsible for transcriptional regulation of target genes. However, recent studies have led to the identification of other kappa B binding proteins such as c-Rel, RelB and p52 (NFKB2/lyt-10) although their role in gene regulation has been less clear. Here, using gel mobility shift assays as well as a highly sensitive DNA-protein crosslinking assay, we provide evidence for the existence of multiple tumor necrosis factor (TNF)- inducible kappa B binding complexes containing various members of the NF-kappa B/Rel family, namely p50 and p65 as well as the c-Rel and p52 oncoproteins. Dimeric complexes containing various combinations of these proteins appear rapidly in nuclei of TNF-alpha-stimulated cells and include, along with a p50-p65 heterodimer, p50-c-Rel, p65-c-Rel, p52-c-Rel and p52-p65 complexes. The presence of multiple inducible complexes containing distinct combinations of NF-kappa B/Rel family members indicate that specific kappa B responsive genes may be regulated in an NF-kappa B subunit-dependent manner. PMID: 8152812 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 400: FEBS Lett. 1994 Apr 4;342(2):115-8. Differential response of NF-kappa B1 p105 and NF-kappa B2 p100 to HTLV-I encoded Tax. Watanabe M, Muramatsu M, Tsuboi A, Arai K. Department of Molecular and Developmental Biology, University of Tokyo, Japan. In a previous study we found that HTLV-I encoded Tax transactivator, which binds to NF-kappa B1 p105, suppresses p105-mediated I kappa B activity, thereby allowing entry of NF-kappa B p65 (RelA) and p50 into the nucleus. In the present report, we compared the effect of Tax on NF-kappa B2 p100, which also binds to Tax, with that of p105 in transfected COS7 cells. While p105 is processed to the DNA binding form, p50, processing of p100 was much less efficient both in the presence and absence of Tax. Both p105 and p100 showed I kappa B activity in sequestering NF-kappa B p65 into the cytoplasm. However, only p105-mediated I kappa B activity, and not that of p100, was inhibited by Tax. Chimeric molecules between p100 and p105 suggested that inefficient processing of p100 can be attributed to the Rel homologous domain, rather than to the ankyrin repeat domain. p100, but not p105, potently suppressed Tax- and p65-induced transaction of a GM-CSF promoter in Jurkat cells. Taken together, these results suggest that p105 and p100 may have distinct effects on Tax-induced transactivation events. PMID: 8143861 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 401: Mol Cell Biol. 1994 Apr;14(4):2593-603. Purification, reconstitution, and I kappa B association of the c-Rel-p65 (RelA) complex, a strong activator of transcription. Hansen SK, Baeuerle PA, Blasi F. University Institute of Microbiology, Copenhagen, Denmark. HeLa cells contain a DNA-binding activity which associates with a kappa B-like DNA element, termed Rel-related protein-binding element (RRBE), localized upstream of the human urokinase promoter. We have purified this activity from the HeLa cell cytosol and have shown that it represents a performed heteromeric complex between p65 (RelA) and c-Rel. Coexpression of c-Rel and p65 (RelA) by in vitro translation formed a DNA-binding complex indistinguishable from purified cellular c-Rel-p65 (RelA) in mobility shift assays. The c-Rel-p65 (RelA) complex was also formed in COS7 cells upon coexpression of c-Rel and p65 (RelA) cDNAs. Cotransfection experiments with COS7 cells, using expression plasmids encoding p50, p65 (RelA), or c-Rel and reporter constructs containing a trimerized RRBE, revealed that c-Rel-p65 (RelA) is a potent activator of the RRBE, giving rise to transcriptional activity higher than that observed with NF-kappa B (p50-p65). In the cytosol, the c-Rel-p65 (RelA) complex existed in a latent, non-DNA-binding form but could be activated by detergent treatment, suggesting that it was associated with an I kappa B protein. Recombinant I kappa B-alpha inhibited the DNA-binding activity of c-Rel-p65 (RelA) via association with either c-Rel or p65 (RelA). Finally, NF-kappa B and c-Rel-p65 (RelA) complexes were found to be differentially expressed and regulated in different cells. The two complexes were present in equimolar amounts in HeLa cells and K562 cells. Stimulation with tetradecanoyl phorbol acetate (TPA) resulted in the nuclear translocation of both NF-kappa B and c-Rel-p65 (RelA) in HeLa cells and of NF-kappa B in HepG2 cells but had no effect on either complex in K562 cells. In addition, TPA stimulation of HepG2 cells induced the expression of a cytosolic latent c-Rel-p65 (RelA) complex which, however, was not translocated to the nucleus. In conclusion, our findings show that c-Rel-p65 (RelA) is an inducible and very potent transcriptional activator which is differentially activated in a cell-type-specific manner. PMID: 8139561 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 402: J Biol Chem. 1994 Feb 18;269(7):4705-8. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. Xie QW, Kashiwabara Y, Nathan C. Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021. The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages. PMID: 7508926 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 403: Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1346-50. Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs. Sun SC, Ganchi PA, Beraud C, Ballard DW, Greene WC. Gladstone Institute of Virology and Immunology, University of California, San Francisco General Hospital 94141-9100. RelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-kappa B complex and contains a high-affinity binding site for its cytoplasmic inhibitor, I kappa B alpha. After cellular activation, I kappa B alpha is rapidly degraded in concert with the induced nuclear translocation of NF-kappa B. The present study demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B alpha in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on I kappa B alpha result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with I kappa B alpha but also with other ankyrin motif-rich proteins including the products of the NF-kappa B2 (p100) and NF-kappa B1 (p105) genes; (ii) in contrast to RelA-I kappa B alpha, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor alpha activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for I kappa B alpha binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with I kappa B alpha, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-kappa B. PMID: 8108414 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 404: J Exp Med. 1994 Feb 1;179(2):503-12. NF-kappa B and I kappa B alpha: an inducible regulatory system in endothelial activation. Read MA, Whitley MZ, Williams AJ, Collins T. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts. Structural analysis of the promoters of several endothelial genes induced at sites of inflammatory or immune responses reveals binding sites for the transcription factor nuclear factor kappa B (NF-kappa B). Endothelial cells express transcripts encoding the p50/p105 and p65 components of NF-kappa B and the rel-related proto-oncogene c-rel; steady state levels of these transcripts are transiently increased by tumor necrosis factor alpha (TNF-alpha). Western blotting revealed that stimulation of endothelial cells with TNF-alpha resulted in nuclear accumulation of the p50 and p65 components of NF-kappa B. Ultraviolet crosslinking and immunoprecipitation demonstrated binding of the p50 and p65 components of NF-kappa B to the E-selectin kappa B site. Endothelial cells express an inhibitor of NF-kappa B activation, I kappa B-alpha (MAD-3). Protein levels of this inhibitor fall rapidly after TNF-alpha stimulation. In parallel, p50 and p65 accumulate in the nucleus and RNA transcript levels for I kappa B-alpha are dramatically upregulated. Recombinant p65 stimulates expression of E-selectin promoter-reporter constructs. I kappa B-alpha inhibits p65 or TNF-alpha-stimulated E-selectin promoter-reporter gene expression in transfected endothelial cells. The NF-kappa B and I kappa B-alpha system may be an inducible regulatory mechanism in endothelial activation. PMID: 7507507 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 405: J Immunol. 1994 Jan 1;152(1):12-21. Visualization of the endogenous NF-kappa B p50 subunit in the nucleus of follicular dendritic cells in germinal centers. Feuillard J, Korner M, Fourcade C, Costa A, Binet JL, Debre P, Raphael M. Departement d'Hematologie, Hopital Pitie Salpetriere, Paris, France. NF-kappa B, a 50 kDa/65 kDa (p50/p65) heterodimer, is a ubiquitous transcription factor involved in the positive regulation of various immune genes. The aim of this study was to determine whether NF-kappa B is related to a particular cell type and/or differentiation step during immunopoiesis. Using in situ hybridization on sections from non HIV hyperplastic lymph nodes, we found that the gene of the 105 kDa precursor of p50 was overexpressed in the light zone of germinal centers, with a network aspect, which suggested the involvement of follicular dendritic cells (FDC). By immunohistochemistry, p50 protein was detected in the cytoplasm and nucleus of FDC, confirming the involvement of FDC. Furthermore, p50 protein was detected in the cytoplasm of all lymphocytes. Thus, we focused our study on isolated FDC clusters from normal tonsils. As showed on tissue sections, we detected the p50 in both cytoplasm and nucleus of FDC. Nuclei of lymphocytes from FDC clusters were negative. We next studied p65 and c-Rel protein expression in FDC clusters. p65 was detected in the cytoplasm of FDC, whereas nuclei were negative. Furthermore, p65 was detected in the nuclei of some lymphocytes. c-Rel protein was detected only in the cytoplasm of lymphocytes and not in the nucleus and cytoplasm of FDC. Our results indicated that, in the context of T cell-dependent B cell immunopoiesis occurring in FDC clusters, p50 is mainly related to FDC with a massive overexpression in the nuclei, whereas p65 is expressed in a scattered manner in the nuclei of lymphocytes and c-Rel protein exclusively in the cytoplasm of lymphocytes from FDC clusters. These results suggested that the two subunits of NF-kappa B and the c-Rel protein have different roles in different cell types during B cell immunopoiesis. PMID: 8254185 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 406: EMBO J. 1993 Dec 15;12(13):5043-9. Promoter analysis of the gene encoding the I kappa B-alpha/MAD3 inhibitor of NF-kappa B: positive regulation by members of the rel/NF-kappa B family. Le Bail O, Schmidt-Ullrich R, Israel A. Unite de Biologie Moleculaire de l'Expression Genique, URA 1149 CNRS, Institut Pasteur, Paris, France. In order to characterize the regulation of the gene encoding the I kappa B-alpha/MAD3 inhibitor of the transcription factor NF-kappa B, we have isolated a human genomic clone and sequenced the promoter of this gene. The MAD3 promoter exhibits a potential TATA element upstream of one of the two major transcription sites, and contains several potential NF-kappa B binding sequences, suggesting that the gene is positively regulated by members of this family. Transfection experiments demonstrate that the MAD3 promoter can be activated by various combinations of members of the rel/NF-kappa B family, as well as by phorbol esters and tumor necrosis factor. Specific deletion of one of the kappa B motifs, located 37 bp upstream of the TATA box, abolishes responses to PMA and TNF. This kappa B motif binds NF-kappa B (p50/relA), p50/c-rel and relA/c-rel heterodimers as well as KBF1 (p50 homodimer). These results help to explain the previously observed transient nature of the NF-kappa B response: following NF-kappa B activation, the expression of the inhibitor is increased, therefore the extent of nuclear translocation of the active complex is reduced, resulting in a decreased activation of its target genes. PMID: 8262046 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 407: Gene. 1993 Nov 15;133(2):237-42. Isolation of the chicken NF-kappa B p65 subunit-encoding cDNA and characterization of its products. Ikeda T, Honjo K, Hirota Y, Onodera T. National Institute of Animal Health, Ibaraki, Japan. NF-kappa B is a heterodimeric transcription factor consisting of subunits of 50 kDa (p50) and 65 kDa (p65). cDNA clones encoding the chicken NF-kappa B p65 subunit were isolated. Sequence analysis showed that chicken p65 is approximately 55% identical to the mouse and human p65 proteins, and contains the Rel homology domain (RHD) in its N-terminal 286 amino acids (aa) and the putative transactivation domain in its C-terminal region. The RHD is particularly highly conserved between the chicken and mammalian p65 proteins. Northern blot hybridization analysis detected the expression of a 2.6-kb transcript of p65 in various organs, with the highest level in spleen. A fusion protein containing the RHD of chicken p65 was found to bind to a consensus kappa B-site in an electrophoretic mobility shift assay (EMSA). This binding was specifically inhibited by the presence of fusion proteins containing the C-terminal ankyrin repeats domain (ARD) of chicken p105, the precursor protein for the p50 subunit. Immunoprecipitation analysis showed that p65 formed a complex(es) with multiple cellular proteins, including p50, p105 and c-Rel in chicken spleen cells. PMID: 7916720 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 408: Oncogene. 1993 Nov;8(11):2889-96. Two novel functions associated with the Rel oncoproteins: DNA replication and cell-specific transcriptional activation. Ishikawa H, Asano M, Kanda T, Kumar S, Gelinas C, Ito Y. Department of Viral Oncology, Kyoto University, Japan. The v-Rel oncoprotein and its cellular homolog c-Rel belong to the Rel/kappa B family of transcription factors. Members of this family share extensive sequence similarity in their N-terminal halves, a region referred to as the Rel Homology Region (RHR), bind to NF-kappa B DNA motifs and form heterodimers with one another. Whereas c-Rel activates transcription of kappa B-linked genes, v-Rel behaves as a dominant-interfering mutant of c-Rel- and kappa B-mediated transcription activation. Here we describe two novel activities of the Rel oncoproteins. One induces kappa B-site dependent stimulation of polyomavirus (Py) DNA replication and maps to the N-terminus of the RHR, a region where no transcription activation function was detected. This activity is common to v-Rel, c-Rel, p52 (p49/lyt10), RelA (p65) and the p50 subunit of NF-kappa B. The second promotes transcriptional activation in undifferentiated F9 cells and maps 3' to the RHR, a region essential for the transforming activity of v-Rel. PMID: 8414493 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 409: Mol Cell Biol. 1993 Nov;13(11):6733-41. Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB. Xu X, Prorock C, Ishikawa H, Maldonado E, Ito Y, Gelinas C. Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5638. Rel family proteins regulate the expression of genes linked to kappa B-binding motifs. Little is known, however, of the mechanism by which they enhance transcription. We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery. Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein (TBP) and transcription factor IIB (TFIIB) in vitro and in vivo. We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene. Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro, these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins. PMID: 8413269 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 410: Nature. 1993 Oct 21;365(6448):767-70. Distinct NF-kappa B/Rel transcription factors are responsible for tissue-specific and inducible gene activation. Lernbecher T, Muller U, Wirth T. Zentrum fur Molekulare Biologie, Heidelberg, Germany. The NF-kappa B/Rel family is a growing class of transcriptional regulators whose members share the conserved Rel-homology domain, involved in specific DNA binding and dimerization. They interact with the regulatory elements of many different genes and are involved in the regulation of lymphoid-specific and inducible transcription. We tested whether these factors could alone activate a gene in transgenic mice. We report here that a minimal promoter containing three copies of a binding site for these proteins allows tissue-specific and inducible transgene activation. In lymphoid tissues constitutive transgene expression correlates with the presence of a constitutively active p50/RelB heterodimer. Other organs that only contain the p50 homodimer do not express the transgene. In contrast to this constitutive activity mediated by p50/RelB, the p50/p65 heterodimer (which is NF-kappa B) could confer inducible transgene activation in embryo fibroblasts. Thus two different members of the NF-kappa B/Rel family of transcriptional activators are involved in tissue-specific and inducible gene activation in transgenic mice. PMID: 7692309 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 411: Mol Cell Biol. 1993 Oct;13(10):6231-40. Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. Hiscott J, Marois J, Garoufalis J, D'Addario M, Roulston A, Kwan I, Pepin N, Lacoste J, Nguyen H, Bensi G, et al. Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada. The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors. PMID: 8413223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 412: Mol Cell Biol. 1993 Oct;13(10):6137-46. NF-kappa B subunit-specific regulation of the interleukin-8 promoter. Kunsch C, Rosen CA. Human Genome Sciences, Rockville, Maryland 20850. Interleukin-8 (IL-8), a chemotactic cytokine for T lymphocytes and neutrophils, is induced in several cell types by a variety of stimuli including the inflammatory cytokines IL-1 and tumor necrosis factor alpha TNF-alpha. Several cis elements, including a binding site for the inducible transcription factor NF-kappa B, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-kappa B subunits to bind to, and activate transcription from, the IL-8 promoter. A nuclear complex was induced in phorbol myristate acetate-treated Jurkat T cells which bound specifically to the kappa B site of the IL-8 promoter and was inhibited by addition of purified I kappa B alpha to the reaction mixture. Only antibody to RelA (p65), but not to NFKB1 (p50), NFKB2 (p50B), c-Rel, or RelB was able to abolish binding, suggesting that RelA is a major component in these kappa B binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the kappa B element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the kappa B/Rel family examined, the IL-8 kappa B site bound only to RelA and to c-Rel and NFKB2 homodimers, but not to NFKB1 homodimers or heterodimers of NFKB1-RelA. Transient transfection analysis demonstrated a kappa B-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-kappa B subunits indicated that RelA and c-Rel, but neither NFKB1 nor heterodimeric NFKB1-RelA, was able to activate transcription from the IL-8 promoter. Furthermore, cotransfection of NFKB1 and RelA, although able to support activation from the human immunodeficiency virus type 1 long terminal repeat, failed to activate expression from the IL-8 promoter. Antisense oligonucleotides to RelA, but not NFKB1, inhibited phorbol myristate acetate-induced IL-8 production in Jurkat T lymphocytes. These data demonstrate the differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a kappa B-regulated promoter in which the classical NF-kappa B complex is unable to activate transcription from the kappa B element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression. PMID: 8413215 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 413: Mol Cell Biol. 1993 Oct;13(10):6089-101. NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule. Scheinman RI, Beg AA, Baldwin AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599. NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappa B p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an I kappa B-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappa B activity distinct from the classic I kappa B pathway. PMID: 8413211 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 414: Mol Gen Genet. 1993 Oct;241(1-2):129-40. Genetic organization of the streptokinase region of the Streptococcus equisimilis H46A chromosome. Mechold U, Steiner K, Vettermann S, Malke H. Institute for Molecular Biology, Jena University, Germany. The complete nucleotide sequences of four genes and one open reading frame (ORF1) adjacent to the streptokinase gene, skc, from Streptococcus equisimilis H46A were determined. These genes are encoded on the opposite DNA strand to skc and are arranged as follows: dexB-abc-lrp-skc-ORF1-rel. The dexB gene, coding for an alpha-glucosidase (M(r) 61,733), and abc, encoding an ABC transporter (M(r) 42,080), are similar to the dexB and msmK genes, respectively, from the multiple sugar metabolism operon of S. mutans. The lrp gene specifies a leucine-rich protein (M(r) 32,302) that has a leucine-zipper motif at its C-terminus. The function of the Lrp protein is not known but appeared to be detrimental when overexpressed in Escherichia coli. Although lrp appears not to be an essential gene, as judged by plasmid insertion mutagenesis, it is conserved in all streptococcal strains carrying a streptokinase gene. The rel gene showed significant homology to the E. coli relA and spoT genes involved in the stringent response to amino acid deprivation. Multiple alignment of the amino acid sequences of Rel (M(r) 83,913), RelA and SpoT revealed 59.4% homology of the primary structures. Northern hybridization analyses of the genes in the skc region showed skc to be transcribed most abundantly. In addition to transcripts for skc, monocistronic mRNAs were detected for all three genes divergently transcribed from skc. Although there was also some read-through transcription from lrp into abc, and from abc into dexB, the transcription pattern suggests a high degree of transcriptional and functional independence not only of skc but also abc and dexB. Prominent structural features in intergenic regions included a static DNA bending locus located upstream and a putative bidirectional transcription terminator downstream of skc. PMID: 8232196 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 415: Mol Cell Biol. 1993 Oct;13(10):6283-9. Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-kappa B subunits in endothelial and epithelial cells. Shu HB, Agranoff AB, Nabel EG, Leung K, Duckett CS, Neish AS, Collins T, Nabel GJ. Department of Internal Medicine, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650. Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two kappa B-like regulatory elements. Because NF-kappa B can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-kappa B family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-kappa B family members to VCAM-1 gene regulation. We show that both kappa B sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-kappa B subunits. At low concentrations, RelA(p65) acted in concert with the approximately 50-kDa product of p105 NF-kappa B, NF-kappa B1(p50), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-kappa B2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-kappa B2(p49)/RelA(p65) with radiolabeled VCAM kappa B site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-kappa B subunits, with optimal response to p49(100)/p65. Analysis of NF-kappa B mRNA in human umbilical vein endothelial cells revealed that nfkb1, nfkb2, and relA NF-kappa B but not c-rel were induced by tumor necrosis factor alpha and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-kappa B regulate VCAM-1 and differentially activate other genes in these cells. PMID: 7692229 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 416: J Virol. 1993 Sep;67(9):5235-46. Chronic human immunodeficiency virus type 1 infection stimulates distinct NF-kappa B/rel DNA binding activities in myelomonoblastic cells. Roulston A, Beauparlant P, Rice N, Hiscott J. Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada. The relationship between human immunodeficiency virus type 1 (HIV-1) infection and the induction of NF-kappa B binding activity was examined in a myeloid cell model of HIV-1 infection derived from the PLB-985 cell line. Chronic infection of PLB-985 cells led to increased monocyte-specific surface marker expression, increased c-fms gene transcription, and morphological alterations consistent with differentiation along the monocytic pathway. PLB-IIIB cells displayed a constitutive NF-kappa B-like binding activity that was distinct from that induced by tumor necrosis factor alpha or phorbol 12-myristate 13-acetate treatment of the parental PLB-985 cell line. This unique DNA binding activity consisted of proteins of 70, 90, and 100 kDa with a high degree of binding specificity for the NF-kappa B site within the PRDII domain of beta interferon. In this report, we characterize the nature of these proteins and demonstrate that binding of these proteins is also induced following Sendai paramyxovirus infection. The 70-kDa protein corresponds to the NF-kappa B RelA (p65) subunit, which is activated in response to an acute paramyxovirus infection or a chronic HIV-1 infection. Virus infection does not appear to alter the amount of RelA (p65) or NFKB1 (p50) but rather affects the capacity of I kappa B alpha to sequester RelA (p65), therefore leading to constitutive levels of RelA DNA binding activity and to increased levels of NF-kappa B-dependent gene activity. The virally induced 90- to 100-kDa proteins have a distinct binding specificity for the PRDII domain and an AT-rich sequence but do not cross-react with NF-kappa B subunit-specific antisera directed against NFKB1 (p105 or p50), NFKB2 (p100 or p52), RelA (p65), or c-rel. DNA binding of the 90- to 100-kDa proteins was not inhibited by recombinant I kappa B alpha/MAD-3 and was resistant to tryptic digestion, suggesting that these proteins may not be NF-kappa B related. Transient cotransfection experiments demonstrated that RelA and NFKB1 expression maximally stimulated HIV-1 LTR- and NF-kappa B-dependent reporter genes; differences in NF-kappa B-like binding activity were also reflected in higher constitutive levels of NF-kappa B-regulated gene expression in HIV-1-infected myeloid cells. PMID: 8394446 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 417: Oncogene. 1993 Sep;8(9):2567-73. I kappa B alpha can localize in the nucleus but shows no direct transactivation potential. Cressman DE, Taub R. Department of Genetics, University of Pennsylvania, School of Medicine, Philadelphia 19104-6145. Although I kappa B is a cytoplasmic inhibitor of NF-kappa B and c-Rel that prevents nuclear translocation of NF-kappa B, some forms of I kappa B have been found in the nucleus. Given that some other proteins with ankyrin-type repeats are transcription factors, we wondered if a nuclear form of I kappa B alpha could itself be a transcriptional activator. We found that Gal4-I kappa B alpha fusion proteins strongly transactivate a Gal4 site-containing promoter in 3T3 fibroblasts. The I kappa B alpha domain responsible for this transactivation is not the acidic domain of I kappa B alpha, but the ankyrin repeat domain which is responsible for protein-protein interactions. To enhance our ability to detect cellular I kappa B alpha by immunofluorescence, we overexpressed the protein in transfected cells, and found that overexpressed I kappa B alpha is largely cytoplasmic in serum-deprived cells, but nuclear in serum-stimulated cells. However, in cell fractionation studies under all treatment conditions, I kappa B alpha appears mainly in cytoplasmic fractions, suggesting that it can rapidly move out of the nucleus through nuclear pores during extract preparation. Using double antibody immunoprecipitations, we found that I kappa B alpha in proliferating cells is strongly associated with RelA(p65). When I kappa B alpha is fused to the Gal4 DNA-binding domain, nuclear Gal4-I kappa B alpha is associated with RelA(p65). Thus, the activation domain of the associated RelA(p65) molecule could account for the ability of Gal4-I kappa B alpha to transactivate the Gal4 promoter. Unlike Bcl-3, an I kappa B which has been recently shown to directly transactivate through kappa B sites when associated with NFKB2 (p52), I kappa B alpha shows no ability to directly transactivate target promoters via its association with RelA(p65). PMID: 8361766 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 418: Mol Cell Biol. 1993 Jul;13(7):3850-9. Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA. Coleman TA, Kunsch C, Maher M, Ruben SM, Rosen CA. Department of Gene Regulation, Roche Institute of Molecular Biology, Nutley, New Jersey 07110. The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins. PMID: 8321192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 419: Nucleic Acids Res. 1993 May 11;21(9):2157-63. GAL4-I kappa B alpha and GAL4-I kappa B gamma activate transcription by different mechanisms. Morin PJ, Subramanian GS, Gilmore TD. Department of Biology, Boston University, MA 02215. I kappa B proteins regulate Rel/NF-kappa B transcription complexes through a direct protein-protein interaction. In addition, we have previously shown that certain I kappa B proteins (I kappa B alpha and I kappa B gamma) can act as activators of transcription when fused to the DNA-binding domain of GAL4. We now show that a mutant chicken I kappa B alpha protein that cannot interact with Rel proteins in vitro did not activate transcription when fused to GAL4 in chicken embryo fibroblasts (CEF) and Saccharomyces cerevisiae, and did not inhibit growth in yeast; in contrast, an I kappa B alpha mutant that can still interact in vitro with Rel proteins activated transcription in both CEF and yeast and inhibited growth in yeast. In CEF, GAL4-I kappa B alpha mediated transcription activation was inhibited by co-transfection with an expression vector for a RelA (p65) protein that contained sequences needed for interaction with I kappa B alpha but that was deleted of its transcription activation domain. Therefore, it appears that GAL4-I kappa B alpha activates transcription by interacting with an endogenous Rel family protein in CEF. In contrast, the activation domain from I kappa B gamma behaved as a genuine acidic activator of transcription and did not inhibit growth when expressed in yeast. Since transcription activation and growth inhibition by GAL4-I kappa B alpha mutants in yeast correlated with their ability to interact with vertebrate Rel proteins, our results suggest that these activities of GAL4-I kappa B alpha are mediated through interaction with a Rel-like protein in yeast, which is important for cell growth. PMID: 8502557 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 420: Mol Cell Biol. 1993 Mar;13(3):1315-22. Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3). Duckett CS, Perkins ND, Kowalik TF, Schmid RM, Huang ES, Baldwin AS Jr, Nabel GJ. Department of Internal Medicine, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650. Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3. PMID: 8441377 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 421: Mol Microbiol. 1992 Aug;6(15):2191-200. Decreasing transcription elongation rate in Escherichia coli exposed to amino acid starvation. Vogel U, Sorensen M, Pedersen S, Jensen KF, Kilstrup M. Institute of Biological Chemistry, University of Copenhagen, Denmark. The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the 'late' probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth-rate. The transcription elongation rate was c. 43 nucleotides s-1 during exponential growth and decreased abruptly to c. 20 nucleotides s-1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lac-promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA' chains were continued into full-length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s-1 in the unperturbed growth phase to approximately 6 as s-1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed. PMID: 1406259 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 422: Res Microbiol. 1991 Feb-Apr;142(2-3):137-40. A calcium-binding protein that may be required for the initiation of chromosome replication in Escherichia coli. Guzman EC, Pritchard RH, Jimenez-Sanchez A. Departamento de Bioquimica y Biologia Molecular y Genetica, Universidad de Extremadura, Badajoz, Spain. Starvation for isoleucine but not for other amino acids in an ilv- strain or the addition of valine in an ilv+ strain inhibits initiation of chromosome and minichromosome replication in stringent (Rel+) Escherichia coli, but it does not inhibit replication in relaxed (relA) mutants (Guzman et al, 1988). From these results, we concluded that, (1) oriC initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation. These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC initiation. We also present evidence that this protein may be a calcium-binding protein located at 73 min in the genetic map. PMID: 1925011 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 423: Biochemistry. 1989 Feb 21;28(4):1814-9. Relationship between guanosine tetraphosphate and accuracy of translation in Salmonella typhimurium. Negre D, Cortay JC, Donini P, Cozzone AJ. Laboratoire de Biologie Moleculaire, Universite de Lyon, Villeurbanne, France. In bacteria a high level of mistranslation is observed in amino acid starved rel-, but not rel+, strains, and mistranslation can be studied qualitatively by means of "stuttering" experiments in two-dimensional protein gels. It has been suggested that the low level of mistranslation that occurs in rel+ strains is assured by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a nucleotide whose intracellular concentration greatly increases in rel+ cells under amino acid starvation. In the present study the relationship between level of ppGpp and mistranslation was analyzed by performing stuttering experiments in amino acid starved bacteria that contained either high or low levels of ppGpp. Three strains of Salmonella typhimurium were used in these experiments: a relA+ hisT+ strain (TA997), a relA+ hisT strain (TA1001), and a relA hisT strain (PD2). These strains were first characterized with respect to macromolecular syntheses and ppGpp levels under exponential growth and under amino acid starvation. Both rel+ strains exhibited stringent control over RNA synthesis. ppGpp accumulated to high levels when TA997 was starved for either of three amino acids. Starvation of TA1001 for histidine did not cause accumulation of ppGpp, whereas starvation for lysine and arginine produced high levels of ppGpp. Extracts from the three strains, obtained either under exponential growth or under amino acid starvation, were then subjected to two-dimensional electrophoretic anaylsis: mistranslation was observed whenever ppGpp was absent. In particular, starvation of TA1001 for histidine resulted in high mistranslation frequencies, while under lysine and arginine starvation mistranslation was undetectable, regardless of whether the cells were rel+ or rel-.(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 2470403 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 424: Genetics. 1987 Jan;115(1):101-6. Recessive lethal mutations and the maintenance of duplication-bearing strains of Dictyostelium discoideum. Welker DL, Williams KL. Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation. PMID: 3557106 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 425: Mol Gen Genet. 1986 Apr;203(1):150-3. Effect of relA function on the replication of plasmid pBR322 in Escherichia coli. Lin-Chao S, Bremer H. Replication of the plasmid pBR322, and the accumulation and life time of its primer transcript, RNAII, and replication inhibitor, RNAI, were measured in an isogenic relA+/relA pair of E. coli strains during exponential growth, or following amino acid starvation, or during treatment with chloramphenicol. (1) The synthesis rates of RNAI and RNAII decreased during inhibition of protein synthesis in either strain, i.e. their promoters are not under stringent control; (2) during amino acid starvation, RNAI and RNAII lifetimes increased in complex, rel-dependent patterns; (3) the changes in RNAI and RNAII synthesis and accumulation had no immediate effect on the rate of plasmid replication; (4) continued plasmid replication requires a protein which is synthesized during amino acid deprivation or treatment with low concentrations of chloramphenicol in relA+, but not in relA bacteria. PMID: 2423847 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 426: J Bacteriol. 1984 May;158(2):746-8. Degradation of aspartate transcarbamylase in Bacillus subtilis is deficient in rel mutants but is not mediated by guanosine polyphosphates. Bond RW, Switzer RL. Degradation of aspartate transcarbamylase in growing and starved Bacillus subtilis was deficient in relA and relC mutants, but these effects were not correlated with differences in the intracellular level of guanosine polyphosphates. PMID: 6427186 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 427: J Biochem (Tokyo). 1984 Mar;95(3):895-7. Accumulation of relA gene-independent ppGpp in Bacillus subtilis vegetative cells upon temperature shift-down. Ikehara K, Okada H, Maeda K, Ogura A, Sugae K. Both Bacillus subtilis BR16S (rel+) and BR16R (relA-) cells accumulated ppGpp after a temperature shift-down from 37 to 0 degree C. This indicates that a ppGpp accumulation system is present in B. subtilis vegetative cells, which is induced upon cold-shock treatment and is mediated by a relA gene-independent product. PMID: 6427205 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 428: J Bacteriol. 1983 Dec;156(3):1099-106. Rates of peptidoglycan turnover and cell growth of Bacillus subtilis are correlated. Cheung HY, Vitkovic L, Freese E. Peptidoglycan turnover was measured by the decrease of trichloroacetic acid-precipitable label in cells labeled with N-acetyl-D-[14C]glucosamine. The rate of turnover was reduced strongly by the inhibition of RNA or protein synthesis and weakly by the inhibition of lipid, peptidoglycan, or DNA synthesis. It increased with the growth rate (which was controlled by the concentration of oxomethylvalerate limiting the intracellular isoleucine supply) to the same degree in stringent (rel+) and isogenic relaxed (relA) strains. In these and all other strains tested, the turnover rate (k) increased with the growth rate (g) according to the equation, k = 0.70 X g1.38, even when the growth rate was systematically altered by changes in the temperature or in the composition of the medium. PMID: 6196348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 429: Genetika. 1983;19(2):217-20. [Functioning of amino acid operons in Escherichia coli strains with an altered transcription and translation apparatus. II. The effect of mutations in genes coding ribosomal protein S5 and translation elongation factor G on the functioning of the ilv operon] [Article in Russian] Gordeev VK, Turkov MI. It has been shown in our previous study that mutations in genes relA, relC and rpsL result in the delay in Escherichia coli ilv operon derepression; the complete inhibition of derepression of the ilv operon is observed in the double mutants having alterations in rpsL and relA or relC genes. At present, some mutations occurring in the fus gene and altering the structure of the translational elongation G factor have been also found to delay derepression of E. coli ilv operon and complete inhibition in fusr and rel double mutants. Phenotypical ile and val auxotrophy is also detected in the double E. coli mutants with spectinomycin resistance mutation in rpsE gene coding for the structure of ribosomal S5 protein and mutations in relA or relC genes. The suggestion of participation of the ilv operon in regulation of other E. coli amino acid operons expression is discussed. PMID: 6339322 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 430: J Bacteriol. 1982 Feb;149(2):635-41. A new relaxed mutant of Bacillus subtilis. Price VL, Gallant JA. A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC. PMID: 6173376 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 431: Mol Gen Genet. 1982;186(2):263-8. The regulation of the ammonia assimilatory enzymes in Rel+ and Rel- strains of Salmonella typhimurium. Sales M, Brenchley JE. The influence of the relA1 mutation on the regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.3), was examined. When cells grown in rich media (either Luria broth or glucose-ammonia plus casamino acids) were transferred to a glucose-ammonia medium, the relA mutant failed to resume growth and did not have the same increase in any of the assimilatory enzyme activities as the rel+ strain. This effect was particularly dramatic for glutamate dehydrogenase, which increased 6-fold in the rel+ strain. Measurements of the guanosine nucleotide concentrations showed that the rel+ strain had a ppGpp concentration about 9 times that of the relA mutant 5 min after the shift to minimal medium. These results are consistent with those for other biosynthetic enzymes and show that the ammonia assimilatory enzymes require a relA product for their synthesis during shift from rich to minimal media. In addition, we examined the response of these strains to a change in nitrogen source. The relA mutant again failed to resume growth after a shift from glucose-ammonia to glucose-arginine medium. Even though the ppGpp concentration did not increase, the rel+ strain grew and increased glutamine synthetase activities about 2-fold. These changes the absence of increased ppGpp levels suggest that some other relA-mediated function is important during this change in nitrogen source. PMID: 6287174 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 432: Biokhimiia. 1981 Jul;46(7):1267-76. [Dependence of threonine operon expression on the relA gene allelic state and the level of guanosine tetraphosphate in E. coli] [Article in Russian] Perel'man BV, Shakulov RS. The expression of threonine operon in Rel+ and Rel- E. coli cells was studied at the transcriptional level. The rel+ genotype and the lack of a specific amino acid--threonine--are necessary for the stimulation of threonine operon transcription. Under these conditions the RNA fraction, which is thr-mRNA, is increased 2-fold. The lack of arginine or histidine in Rel+ strain does not lead to derepression of the threonine operon. It is shown that in a cell-free system 0.1 mM ppGpp stimulate the synthesis of thr-mRNA on phage DNA lambda dthr and plasmid DNA PYN1107, containing total threonine operon 1.5--2-fold. It is assumed that ppGpp stimulates the initiation of transcription. Studies on the strain carrying spoT- mutation, which significantly lowers the rate of ppGpp degradation and results in suppression of rel- phenotype, revealed positive correlation between the intracellular level of ppGpp and the thr-mRNA fraction in the total transcript. PMID: 6791707 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 433: J Bacteriol. 1981 Jan;145(1):641-3. Mechanism of the rel defect in beta-galactosidase synthesis. Foley D, Dennis P, Gallant J. Relaxed (relA) mutants of Escherichia coli are defective in beta-galactosidase synthesis during amino acid limitation. We show here that this defect comprises both a transcriptional component and a translational component. PMID: 6780525 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 434: Mol Gen Genet. 1981;181(4):476-83. Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae. Riesenberg D, Kari C. A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way. The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F'-factor carrying the relA+ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F'-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent. The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA+ allele, showed the wild-type level of nitrogenase activity under the same conditions. PMID: 6267422 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 435: Biokhimiia. 1980 Oct;45(10):1788-96. [Characteristics of intracellular proteolysis in the cells of Bacillus subtilis] [Article in Russian] Belitskii BR, Shakulov RS. The rate of total protein degradation down to acid-soluble products in the B. subtilis cells growing on a minimal medium is about 4--5% per hour. Under amino acid deficiency the rate of proteolysis depends on the allelic state of the relA gene, so that in the rel+ cells it increases two-fold, while in the rel- cells it remains low. Elimination of NH4+, PO43- and Mg2+ from the culture medium or an addition of NaN3 (8 mM) or 2,4-dinitrophenol (2 mM) results in 1.5--2.0-fold stimulation of proteolysis independently of the relA gene. In all cases studied the rate of proteolysis decreases after addition of chloramphenicol (100 micrograms/ml). It is proposed that chloramphenicol decreases the intracellular concentration of ppGpp, which is believed to exert pleiotropic alterations of cellular metabolism under condition of growth limitation. Quite different is the case of degradation of anomalous proteins synthesized in the presence of the lysine analog--S-2-aminoethylcystein. Degradation of anomalous proteins proceeds very rapidly (about 70% per hour); chloramphenicol (100 micrograms/ml) decreases the rate of proteolysis only two-fold. It was found that tetracycline (100 micrograms/ml) effectively inhibits the degradation of anomalous proteins. This activity of tetracycline was not observed in the presence of 50 mM of Mg2+ and seems to be dependent on the capacity of the antibiotic to form complexes with bivalent cations. These results reveal common features of control of proteolysis in the cells of B. subtilis and E. coli. PMID: 6786368 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 436: J Bacteriol. 1980 Apr;142(1):362-5. Regulation of membrane phospholipid syntheesis in Escherichia coli during temperature up-shift. Kainuma-Kuroda R, Goelz S, Cronan JE Jr. The synthesis of membrane phospholipids and that of stable ribonucleic acid were inhibited during temperature up-shift of both rel+ and relA strains of Escherichia coli. The kinetics of the inhibition of the synthesis of both molecules were correlated with the kinetics of guanosine 5'-diphosphate-3'-diphosphate synthesis. Metabolic down-shift experiments gave similar results. PMID: 6154689 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 437: Mol Gen Genet. 1980;178(2):271-9. Genetics and physiology of the rel system of Bacillus subtilis. Smith I, Paress P, Cabane K, Dubnau E. Stringent factor (ATP:GTP-3' pyrophosphotransferase) has been purified from wild type Bacillus subtilis and it has been shown that guanosine tetra- and pentaphosphate (ppGpp and pppGpp) are synthesized in vitro in the presence of ribosomes, unacylated tRNA and its specific codon, as has been demonstrated in Escherichia coli. relA, the genetic determinant for the stringent factor, has been mapped on the B. subtilis chromosome by transduction and is found between aroD and leu. The relC locus, defined by mutations which were originally selected by resistance to thiostrepton, has been mapped adjacent to spoOH in the order cysA, spoOH, relC, rif. Sringent factor and ribosomes are functional for the in vitro synthesis of (p)ppGpp in early stages of sporulation (up to at least 4 h). This contradicts the findings of other laboratories. PMID: 6248722 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 438: Mol Gen Genet. 1980;179(2):319-25. The relA locus and the regulation of lysine biosynthesis in Escherichia coli. Patte JC, Morand P, Boy E, Richaud C, Borne F. The allelic state of relA influences the phenotype of Escherichia coli strains carrying the lysA22 mutation:lysA22 relA strains are Lys- where lysA22 relA+ strains grow (slowly) in the absence of lysine. This physiological effect has been related to an effect of the expression of the relA locus on the regulation of lysine biosynthesis. The fully derepressed levels of some lysine enzymes (aspartokinase III, aspartic semialdehyde dehydrogenase, didhydrodipicolinate reductase) are observed under lysine limitation only in rel+ strains. And the induction of DAP-decarboxylase by DAP is much higher in rel+ than in rel- strains when an amino acid limitation of growth is also realised. These results are in agreement with the hypothesis of Stephens et al. (1975) on a possible role of the stringent regulation as a general signal for amino acid deficiency. PMID: 6110161 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 439: Arch Microbiol. 1979 May;121(2):181-6. Relaxed mutants of Serratia marcescens SM-6. Biochemical traits and relevance of the rel+ allele for the formation of exoenzymes. Bohne L, Winkler U. Serratia marcescens SM-6 when starved for a required amino acid stops synthesizing protein and RNA and accumulates two nucleotides which co-chromatograph with ppGpp and pppGpp. These features are characteristic of bacterial strains with stringent RNA control (rel+). Two independent mutants were isolated which resemble relaxed (relA) mutants of Escherichia coli; they continue to synthesize RNA and accumulate neither ppGpp nor pppGpp when deprived of the required amino acid. The extracellular enzyme activities (nuclease, protease, lipase) of the relaxed mutants are about the same as those of the parental stringent strain when studied under standard growth conditions. Exoenzyme-deficient (nuc;prt) and exoenzyme-hyperproducing (nucsu) mutants were isolated from both stringent and relaxed strains of S. marcencens SM-6 and no change of the cellular ability to form ppGpp and pppGpp could be observed. From these results it appears that the formation of exoenzymes of S. marcescens SM-6 is independent of stringent/relaxed RNA control. PMID: 384954 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 440: J Bacteriol. 1978 Sep;135(3):766-74. Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli. Ishiguro EE, Ramey WD. The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation. PMID: 357424 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 441: Can J Microbiol. 1978 Jun;24(6):761-4. Effect of amino acid deprivation and chloramphenicol treatment on cell sizes of rel+ and relA- strains of Escherichia coli. Ishiguro EE, Ramey WD. The effects of inhibition of protein synthesis on the cell size distributions of rel+ and relA- derivatives of Escherichia coli K-12 were determined. Amino acid deprivation resulted in a reduction in the cell sizes of rel+ strains but not of relA- strains. Treatment with chloramphenicol (CAM) did not alter the size distributions of either rel+ or relA- strains except when they were rel+ dap-. CAM treatment of rel+ dap- strains resulted in an increase in cell size. It is proposed that these results reflect differences in the structures of the cell envelopes of rel+ and relA- bacteria. PMID: 352500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 442: Biokhimiia. 1978 Jan;43(1):180-3. [Translation of poly U by ribosomes from rel+ and rel- E. coli strains] [Article in Russian] Drobyshev VI, Shakulov RS. A translation of polyU in a cell-free system from CP78 (relA+) and CP79 (relA-) E. coli strains is investigated. The strains studied no not differ in misreading at Mg2+ concentration of 20 mM and concentrations of 16 mM (optimal for phenylalanine incorporation) and 18-22 mM (optimal for leucine incorporation) respectively. It is found that leucine incorporation increases similarly in both strains under conditions simulating an amino acid deficience (in phenylalanine-free incubation medium): the ratio leucine(-phenylalanine)/leucine (+phenylalanine) is 3.5-4 at a concentration of enzymatic fraction protein being 15-30 mkg per example, and it is 2 st a concentration of enzymatic fraction of 60-180 mkg of protein. It is suggested that differences in activities of a number of enzymes under amino acid starvation in Rel+ and Rel- cells are not due to differences in the precision of mRNA translation, but depend on the activity of respective genes. PMID: 341996 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 443: Proc Natl Acad Sci U S A. 1977 Dec;74(12):5529-33. In vitro degradation of guanosine 5'-diphosphate, 3'-diphosphate. Sy J. The degradation of guanosine 5'-diphosphate,3'-diphosphate (ppGpp) by the "crude" ribosomal fraction of Escherichia coli CP78 (rel+, spoT+) was demonstrated and characterized. When the 3'-pyrophosphoryl group of ppGpp was hydrolyzed, the primary degradation product was 5'-GDP. Phosphorylation of ppGpp to guanosine 5'-triphosphate,3'-diphosphate (pppGpp) prior to degradation was not necessary. The degradation process required Mn2+ and was inhibited by EDTA. Levallorphan, an inhibitor of in vivo ppGpp degradation, also inhibited ppGpp degradation by the crude ribosome. Thiostrepton and tetracycline did not have any inhibitory effect, indicating that the reaction is not a reversal of pyrophosphorylation catalyzed by the stringent factor/ribosome complex. Crude ribosome fractions from E. coli NF161 and NF162, both spoT-, contained little degrading activity, but similar fractions of E. coli CP79, a relA- and spoT+ strain, contained ppGpp degrading activity. PMID: 414222 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 444: J Bacteriol. 1977 Oct;132(1):352-5. Effect of the relA gene on derepression of amino acid biosynthetic enzymes in growing Escherichia coli depends on the pathway being derepressed. Furano AV, Wittel FP. Derepression of an enzyme in the arginine biosynthetic pathway, but not of an enzyme in the tryptophan biosynthetic pathway, is inhibited during the stringent response produced by a partial deprivation of valyl transfer ribonucleic acid in a rel+ strain. In contrast, derepression of the tryptophan biosynthetic enzyme, but not of the arginine biosynthetic enzyme, was inhibited during the relaxed response produced in an isogenic relA strain by the partial deprivation of valyl transfer ribonucleic acid. PMID: 334732 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 445: J Bacteriol. 1977 Apr;130(1):114-7. relA gene control of the synthesis of lipid A fatty acyl moieties. Spencer A, Muller E, Cronan JE Jr, Gross TA. The incorporation of [14C]acetate into the fatty acid moieties of lipid A was measured during amino acid starvation of rel+ and relA strains of Escherichia coli K-12. The synthesis of the beta-hydroxymyristate and other fatty acid moieties was inhibited two- to fourfold in rel+ strains, whereas no inhibition was observed in relA strains. The fatty acid compositions of the phospholipids synthesized after amino acid starvation or rel+ and relA strains were also determined. PMID: 323219 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 446: J Biol Chem. 1977 Mar 25;252(6):2154-7. The elongation factor Tu coded by the tufA gene of Escherichia coli K-12 is almost identical to that coded by the tufB gene. Furano AV. Radioactive elongation factor Tu coded by either the tufA or the tufB gene of Escherichia coli K-12 was isolated from cells incubated with a mixture of radioactive amino acids after infection with the defective lambda phage particles that carry either of these genes. Two-dimensional chromatographic analyses of tryptic digests of the tufB gene product revealed about 50 radioactive spots. These same spots plus an additional one were also found in tryptic digests of the tufA gene product. Furthermore, these peptide maps are qualitatively the same as those of the elongation factor Tu obtained from two separate isolates of uninfected E. coli K-12 or from rel+ and relA strains of E. coli B. Because the number of spots recovered is consistent with the number of trypsin-sensitive sites, these analyses indicate that the tufA and tufB genes have not significantly diverged from each other. PMID: 321450 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 447: J Bacteriol. 1977 Jan;129(1):564-6. Role of the rel gene product in the control of cyclic adenosine 3',5'-monophosphate accumulation. Braedt G, Gallant J. The presence of a relA mutant allele affects the kinetics of cyclic adenosine 3',5'-monophosphate accumulation during downshift from glucose to succinate. The nucleotide accumulates at the normal rate early in the downshift transition but continues to accumulate for a longer time in the relA mutant, leading to a two- to threefold excess by the end of the diauxic lag. Evidence is presented that this effect occurs independently of the accumulation of ppGpp. PMID: 187574 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 448: J Bacteriol. 1976 Sep;127(3):1119-26. Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12. Ishiguro EE, Ramey WD. [3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli. PMID: 783130 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 449: Biochemistry. 1976 Jun 15;15(12):2546-50. Regulation of membrane phospholipid synthesis by the relA gene: dependence on ppGpp levels. Nunn WD, Cronan JE Jr. A series of experiments using a pair of isogenic rel+ strains of Escherichia coli differing only in the spoT locus has demonstrated a quantitative correlation between the inhibition of phospholipid synthesis and the intracellular level of ppGpp. The conditions examined were (1) amino acid starvation; (2) release from amino acid starvation; and (3) balanced growth. We also have been shown the presence of a third gene (in addition of relA and spoT) concerned with ppGpp metabolism and have been found that the level of ppGpp during amino acid starvation is unaffected by an increase in the dosage of the relA gene. PMID: 779827 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 450: Cell. 1976 May;8(1):115-22. Effect of the RelA gene on the synthesis of individual proteins in vivo. Furano AV, Wittel FP. We have shown that the relA gene can affect the rates of synthesis of many nonribosomal proteins in E. coli. We used rel+ and relA strains that contain a temperature-sensitive valyl tRNA synthetase. Upon transfer from a permissive temperature (30degreesC) to a semi-resitrictive one (36.5degreesC), these strains continue to grow, although undergoing a partial deprivation of valyl tRNA, In the rel+ strain, the concentration of ppGpp increases immediately, and the accumulation of RNA ceases abruptly but temporarily. In contrast, in the relA strain, the concentration of ppGpp falls, whereas the rate of accumulation of RNA increases. As judged by gel electrophoresis, the rates at which individual polypeptides are synthesized by the strains after their transfer to 36.5degreesC depend to a large extent upon the allelic state of the relA gene. In both strains, the concentration of ppGpp changes soon enough to have altered the synthesis of some of the proteins by affecting the transcription of their genes. PMID: 782722 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------