1: Histol Histopathol. 2006 Jan;21(1):69-80. The NF-kappaB-mediated control of ROS and JNK signaling. Bubici C, Papa S, Pham CG, Zazzeroni F, Franzoso G. The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL 60637, USA. NF-kappaB/Rel transcription factors are best known for their roles in innate and adaptive immunity and inflammation. They also play a central role in promoting cell survival. This latter activity of NF-kappaB antagonizes programmed cell death (PCD) induced by the proinflammatory cytokine tumor necrosis factor (TNF)alpha and plays an important role in immunity, lymphopoiesis, osteogenesis, tumorigenesis and radio- and chemoresistance in cancer. With regard to TNFalpha, the NF-kappaB-mediated inhibition of PCD seems to involve an attenuation of the c-Jun-N-terminal kinase (JNK) cascade mediated through the induction of select downstream targets such as the caspase inhibitor XIAP, the zinc-finger protein A20, and the inhibitor of the MKK7/JNKK2 kinase, Gadd45beta/Myd118. Notably, NF-kappaB also blunts accumulation of reactive oxygen species (ROS), which themselves are pivotal elements for induction of PCD by TNFalpha, and this suppression of ROS formation mediates an additional protective activity recently ascribed to NF-kappaB. The antioxidant activity of NF-kappaB has been shown to depend upon upregulation of both Ferritin heavy chain (FHC)--a component of Ferritin, the primary iron-storage protein complex found in cells--and of the mitochondrial enzyme Mn++ superoxide dismutase (Mn-SOD). Indeed, the inductions of Mn-SOD and FHC represent another important means through which NF-kappaB controls proapoptotic JNK signaling triggered by TNFalpha. These findings might enable the development of new, more targeted approaches to treatment of diseases sustained by a deregulated activity of NF-kappaB, including some cancers and chronic inflammatory conditions. PMID: 16267788 [PubMed - in process] --------------------------------------------------------------- 2: Oncogene. 2005 Oct 17; [Epub ahead of print] LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice. Thornburg NJ, Kulwichit W, Edwards RH, Shair KH, Bendt KM, Raab-Traub N. 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.Oncogene advance online publication, 17 October 2005; doi:10.1038/sj.onc.1209023. PMID: 16247482 [PubMed - as supplied by publisher] --------------------------------------------------------------- 3: J Ethnopharmacol. 2005 Oct 31;102(1):95-101. The immunosuppressive effect of Buchang-tang through inhibition of mitogen-activated protein kinase and nuclear factor activation in MOLT-4 cells. Shin HY, Shin TY, An NH, Kim HR, Chae HJ, Kim YK, Um JY, Hong SH, Kim HM. College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, South Korea; College of Pharmacy, Wonkwang University, Iksan, Jeonbuk 570-749, South Korea. Buchang-tang (BCT) has been known to suppress inflammatory and autoimmune responses. Accordingly, BCT has been clinically used in Korea as an immunomodulatory oriental medicine. Here, we report on the mechanism of action of BCT in activated MOLT-4 cells by determining the affected signaling pathways. BCT inhibits extracellular signal-regulated kinases (ERK)l/2 and p38 activation but does not interfere with phosphorylation of other mitogen-activated protein kinases, c-Jun NH(2)-terminal kinases 1/2 in MOLT-4 cells. The nuclear localization of nuclear factor of activated T cells 2 (NFATc) was blocked by BCT. Also, degradation of inhibitor kappaB-alpha and transactivation by nuclear factor-kappa B (NF-kappaB)/Rel A were impaired. Furthermore, interlukin (IL)-2 mRNA and protein levels were significantly diminished by BCT treatment. Our data indicate that BCT inhibits ERK1/2, p38 activation, nuclear translocation of NFATc, and NF-kappaB, resulting in diminished secretion of IL-2. PMID: 16039080 [PubMed - in process] --------------------------------------------------------------- 4: World J Gastroenterol. 2005 Jul 28;11(28):4337-43. Effect of NS398 on metastasis-associated gene expression in a human colon cancer cell line. Gao XQ, Han JX, Huang HY, Song B, Zhu B, Song CZ. Key Laboratory of Ministry of Public health for Biotech-Drug, Shandong Medicinal and Biological Center, Shandong Academy of Medical Sciences, 89 Jingshi Road, Jinan 250062, Shandong Province, China. AIM: To investigate the effect of NS398 on the metastasis-associated gene expression in LoVo colorectal cancer cells. METHODS: LoVo cells were treated with NS398 at the concentration of 100 micromol/L for 24 and 48 h respectively. Total RNA was extracted with TRIzol reagents and reverse transcribed with Superscript II and hybridized with cDNA microarray (containing oncogenes, tumor suppressor genes, signal transduction molecules, adhesive molecules, growth factors, and ESTs) fabricated in our laboratory. After normalization, the ratio of gene expression of NS398 treated to untreated LoVo cells was either 2-fold up or 0.5-fold down was defined as the differentially expressed genes. Semi-quantitative RT-PCR was used to validate the microarray results. RESULTS: Among the 447 metastasis-associated genes, 9 genes were upregulated and 8 genes were downregulated in LoVo cells treated with NS398 for 24 h compared to untreated cells. While 31 genes were upregulated and 14 genes were downregulated in LoVo cells treated with NS398 for 48 h. IGFBP-5, PAI-2, JUN, REL, BRCA1, and BRCA2 might be the new targets of NS398 in treatment of colorectal cancer. CONCLUSION: NS398 might exert its anti-metastasis effect on colorectal cancer by affecting several metastasis-associated gene expression. PMID: 16038031 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Biol Pharm Bull. 2005 Jul;28(7):1177-82. The immunosuppressive effect of Gamisanghyulyunbueum through inhibition of mitogen-activated protein kinase and nuclear factor activation in MOLT-4 cells. Shin HY, Jeong HJ, Na HJ, Kim HJ, Moon G, Shin TY, Yang DC, Hong SH, Kim HM. College of Oriental Medicine, Kyung Hee University, Seoul, South Korea. Gamisanghyulyunbueum (GSHYBE) has been used clinically to treat skin related disease in South Korea. We investigated GSHYBE-mediated changes in downstream T cell signal transduction. To determine the mechanism of inhibition, we have studied many of the major pathways in phytohemagglutinin (PHA)-activated T cell. We show that among the mitogen-activated protein kinase family activation of phosphorylation of extra cellular signal-regulated kinase 1/2 (ERK1/2, p44/42) and p38, but not c-jun NH2-terminal kinase is inhibited. In activated MOLT-4 cells, the nuclear localization of nuclear factor of activated T cells (NFATc) was blocked by GSHYBE (1 mg/ml). Also, degradation of inhibitor kappaB-alpha and transactivation by nuclear factor-kappaB (NF-kappaB)/Rel A were impaired by GSHYBE (1 mg/ml). Furthermore, interlukin (IL)-2, IL-4 and Interferen (IFN)-gamma secretion by PHA activated MOLT-4 cells and peripheral blood mononuclear cells (PBMC) were significantly diminishes following GSHYBE treatment (1 mg/ml). Also, oral administration of GSHYBE inhibited IL-2 secretion in skin allergic reaction. In conclusion, our data indicate that GSHYBE treatment of T cells inhibits ERK1/2 and p38 activation and nuclear translocation of NFATc, NF-kappaB, resulting in diminished secretion of IL-2. PMID: 15997093 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Synapse. 2005 Sep 1;57(3):179-81. Picomolar concentrations of hibernation induction delta opioid peptide [D-Ala2,D-Leu5]enkephalin increase the nerve growth factor in NG-108 cells. Tsai SY, Hayashi T, Su TP. Cellular Pathobiology Unit/DPS/CNRB, IRP, National Institute on Drug Abuse, NIH/DHHS, Triad Room 3304, 333 Cassell Drive, Baltimore, Maryland 21224, USA. The delta opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) has been shown to be a neuroprotective agent via mechanisms that are not totally understood. We previously demonstrated that the i.p. injection of DADLE in mice causes an increase of nerve growth factor (NGF) in the brain. To further clarify the NGF-increasing action of DADLE, we examined here the NGF-increasing effect of DADLE in vitro, using cultured NG-108 cells. DADLE dose-dependently increases the immunoreactive level of NGF in NG-108 cells in a bell-shape manner, with the effective DADLE concentrations in the picomolar range (0.01-100 pM). Also, DADLE at 1 pM selectively increases c-Jun and c-Fos, but not c-Rel. These results indicate that DADLE is one of the most potent agents in increasing the NGF in the biological system and that this action of DADLE involves selective increases of c-Jun and c-Fos, transcription factors that promote the NGF expression. PMID: 15945062 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Mol Carcinog. 2005 Jun;43(2):108-16. Differential inhibition of UVB-induced AP-1 and NF-kappaB transactivation by components of the jun bZIP domain. Cooper S, Ranger-Moore J, Bowden TG. Arizona Cancer Center, Tucson, Arizona 85724, USA. Potential targets for chemoprevention of nonmelanoma skin cancer include UV-induced nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activation in keratinocytes. Inhibition of both these ultraviolet light B (UVB)-induced transcription factors has been shown with the dominant-negative c-jun mutant, TAM67; however, its mechanism of action has not yet been determined. Here we demonstrated that transient transfection of a mouse keratinocyte cell line (308) with a dominant-negative phosphorylation mutant of c-Jun before exposure to 250 J/m(2) UVB inhibits transactivation mediated by both AP-1 and NF-kappaB transcription factors to levels below those of UVB exposed controls. Through the utilization of immunoprecipitation techniques, protein-protein interactions between NF-kappaB family members IkappaBalpha, IkappaBbeta, p50, and p65 (Rel-A) were identified with an Xpress tagged dominant-negative c-Jun (TAM67) protein. Expression of the leucine zipper domain of the TAM67 protein inhibited UVB-induced NF-kappaB transactivation but not AP-1 transactivation. Expression of the bZIP domain of the TAM67 protein was able to inhibit transactivation mediated by both transcription factors. These data demonstrate that TAM67 is able to inhibit two significant UVB-induced molecular targets AP-1 and NF-kappaB, and that the inhibition of these two transcription factor families is potentially due to protein-protein interactions between different regions of the dominant-negative c-Jun protein. PMID: 15791649 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Cell Cycle. 2004 Dec;3(12):1524-9. Epub 2004 Dec 19. NF-kappaB and JNK: an intricate affair. Bubici C, Papa S, Pham CG, Zazzeroni F, Franzoso G. The Ben May Institute for Cancer Research, The Universityof Chicago, Chicago, Illinois 60637, USA. NF-kappaB/Rel transcription factors block apoptosis or programmed cell death (PCD) induced by tumor necrosis factor (TNF) alpha. The antiapoptotic activity of NF-kappaB is also crucial for immunity, lymphocyte development, tumorigenesis, and cancer chemoresistance. With respect to TNFalpha, the NF-kappaB-mediated suppression of apoptosis involves inhibition of the c-Jun-N-terminal kinase (JNK) cascade. This inhibitory activity of NF-kappaB depends upon transcriptional upregulation of blockers of the JNK cascade such as the caspase inhibitor XIAP, the zinc-finger protein A20, and the inhibitor of the MKK7/JNKK2 kinase Gadd45beta/Myd118. Moreover, NF-kappaB blunts accumulation of reactive oxygen species (ROS) induced by TNFalpha, and this antioxidant effect of NF-kappaB is also critical for inhibition of TNFalpha-induced JNK activation. Suppression of ROS by NF-kappaB is mediated by Ferritin heavy chain (FHC)--the primary iron-storage mechanism in cells--and possibly, by the mitochondrial enzyme Mn++ superoxide dismutase (Mn-SOD). Thus, induction of FHC and Mn-SOD represents an additional, indirect means by which NF-kappaB controls proapoptotic JNK signaling. These findings identify potential new targets for anti-inflammatory and anti-cancer therapy. PMID: 15611622 [PubMed - in process] --------------------------------------------------------------- 9: J Biol Chem. 2005 Mar 4;280(9):8617-27. Epub 2004 Dec 15. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: the requirement of extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and NF-kappaB pathways for the expression of proinflammatory cytokines and nitric oxide. Zhu J, Krishnegowda G, Gowda DC. Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-0850, USA. The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis by eliciting the production of proinflammatory cytokines and nitric oxide by the host innate immune system. In this study we demonstrate that the parasite GPIs can effectively induce the production of TNF-alpha at 5-20 nm concentrations in interferon-gamma-primed monocytes and macrophages. The potency of the parasite GPIs activity is physiologically relevant to their ability to contribute to severe malaria pathogenesis. More importantly, we investigated the requirement of the extracellular signal-regulated kinase (ERK)-, c-Jun N-terminal kinase (JNK)-, p38-, and NF-kappaB-signaling pathways that are activated in response to P. falciparum GPIs through toll-like receptor-mediated recognition (Krishnegowda, G., Hajjar, A. M., Zhu J. Z., Douglass, E. J., Uematsu, S., Akira, S., Wood, A. S., and Gowda, D. C. (2005) J. Biol. Chem. 280, 8606-8616) for the proinflammatory responses by macrophages. The data conclusively show that the production of TNF-alpha, interleukin (IL)-12, IL-6, and nitric oxide by macrophages stimulated with parasite GPIs is critically dependent on the NF-kappaB and JNK pathways. NF-kappaB1 is essential for IL-6 and IL-12 production but not for TNF-alpha and nitric oxide, whereas NF-kappaB/c-Rel appears to be important for all four proinflammatory mediators. JNK1 and JNK2 are functionally redundant for the expression of TNF-alpha, IL-6, and nitric oxide, whereas JNK2 but not JNK1 is essential for IL-12 production. The ERK signaling pathway is not involved in TNF-alpha and nitric oxide production, but, interestingly, negatively regulates the expression of IL-6 and IL-12. Furthermore, p38 is critical for the production of IL-6 and IL-12 but is only marginally required for the production of TNF-alpha and nitric oxide. Thus, our data define the differential requirement of the downstream signaling molecules for the production of key proinflammatory cytokines and nitric oxide by macrophages in response to P. falciparum GPI stimuli. The data have important implications for the development of therapeutics for malaria treatment. PMID: 15611045 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Eur J Biochem. 2004 Sep;271(18):3693-703. Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-beta gene promoter. Yang H, Ma G, Lin CH, Orr M, Wathelet MG. Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576, USA. The interferon-beta promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus-infected cells the transcription factors ATF-2, c-Jun, interferon regulatory factor (IRF)-3, IRF-7 and NF-kappaB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP-1, IRF and Rel proteins fail to activate the interferon-beta gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF-3 or IRF-7, but not IRF-1, was sufficient to allow virus-dependent activation of the interferon-beta promoter. Moreover, the activity of IRF-3 and IRF-7 was strongly affected by promoter context, with IRF-7 preferentially being recruited to the natural interferon-beta promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF-3 and IRF-7 but not IRF-1, and was strongly dependent on the presence of p300/CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon-beta gene depends on a unique promoter context and on the role played by coactivators as architectural factors. Copyright 2004 FEBS PMID: 15355347 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Clin Exp Immunol. 2004 Aug;137(2):329-40. Disruption of MAP kinase activation and nuclear factor binding to the IL-12 p40 promoter in HIV-infected myeloid cells. Chambers KA, Parks RJ, Angel JB. Molecular Medicine Program, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada. Progressive immunodeficiency in HIV infection is paralleled by a decrease in IL-12 production, a cytokine crucial for cellular immune function. Here we examine the molecular mechanisms by which HIV infection suppresses IL-12 p40 expression. HIV infection of THP-1 myeloid cells resulted in decreased LPS-induced nuclear factor binding to the NF-kappaB, AP-1, and Sp1 sites of the IL-12 p40 promoter. By site-directed mutagenesis we determined that each of these sites was necessary for transcriptional activation of the IL-12 p40 promoter. Binding of NF-kappaB p50, c-Rel, p65, Sp1, Sp3, c-Fos, and c-Jun proteins to their cognate nuclear factor binding sites was somewhat impaired by HV infection, although a role for other as yet unidentified factors cannot be dismissed. The cellular levels of these transcription factors were unaffected by HIV infection, with the exception of a decrease in expression of NF-kappaB p65, consistent with the observed decrease in its binding to the IL-12 p40 promoter following HIV infection. Analysis of regulation of upstream LPS-induced MAP kinases demonstrated impaired phosphorylation of JNK and p38 MAPK, and suppressed phosphorylation and degradation of IkappaBalpha following HIV infection. These results suggest that alterations in nuclear factor binding to numerous sites in the IL-12 p40 promoter, together may contribute to the suppression in IL-12 p40 transcription previously reported. These effects on nuclear factor binding may be a direct effect of HIV infection on the IL-12 p40 promoter, or may occur indirectly as a consequence of altered MAP kinase activation. PMID: 15270850 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: 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] --------------------------------------------------------------- 13: J Biol Chem. 2004 Jun 25;279(26):27199-210. Epub 2004 Apr 26. Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis. Wickremasinghe MI, Thomas LH, O'Kane CM, Uddin J, Friedland JS. Department of Infectious Diseases, Imperial College, Hammersmith Campus, London W12 0NN, United Kingdom. CCL5 (or RANTES (regulated upon activation, normal T cell expressed and secreted)) recruits T lymphocytes and monocytes. The source and regulation of CCL5 in pulmonary tuberculosis are unclear. Infection of the human alveolar epithelial cell line (A549) by Mycobacterium tuberculosis caused no CCL5 secretion and little monocyte secretion. Conditioned medium from tuberculosis-infected human monocytes (CoMTB) stimulated significant CCL5 secretion from A549 cells and from primary alveolar, but not upper airway, epithelial cells. Differential responsiveness of small airway and normal human bronchial epithelial cells to CoMTB but not to conditioned medium from unstimulated human monocytes was specific to CCL5 and not to CXCL8. CoMTB induced CCL5 mRNA accumulation in A549 cells and induced nuclear translocation of nuclear factor kappaB (NFkappaB) subunits p50, p65, and c-rel at 1 h; nuclear binding of activator protein (AP)-1 (c-Fos, FosB, and c-Jun) at 4-8 h; and binding of NF-interleukin (IL)-6 at 24 h. CCL5 promoter-reporter analysis using deletion and site-specific mutagenesis constructs demonstrated a key role for AP-1, NF-IL-6, and NFkappaB in driving CoMTB-induced promoter activity. The IL-1 receptor antagonist inhibited A549 and small airway epithelial cell CCL5 secretion, gene expression, and promoter activity. CoMTB contained IL-1beta, and recombinant IL-1beta reproduced CoMTB effects. Monocyte alveolar, but not upper airway, epithelial cell networks in pulmonary tuberculosis cause AP-1-, NF-IL-6-, and NFkappaB-dependent CCL5 secretion. IL-1beta is the critical regulator of tuberculosis-stimulated CCL5 secretion in the lung. PMID: 15117956 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Nat Cell Biol. 2004 Feb;6(2):146-53. Epub 2004 Jan 25. Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Papa S, Zazzeroni F, Bubici C, Jayawardena S, Alvarez K, Matsuda S, Nguyen DU, Pham CG, Nelsbach AH, Melis T, De Smaele E, Tang WJ, D'Adamio L, Franzoso G. The Gwen Knapp Center for Lupus and Immunology Research, and The Ben May Institute for Cancer Research, The University of Chicago, 924 East 57th Street, Chicago, IL 60637, USA. NF-kappa B/Rel transcription factors control apoptosis, also known as programmed cell death. This control is crucial for oncogenesis, cancer chemo-resistance and for antagonizing tumour necrosis factor alpha (TNFalpha)-induced killing. With regard to TNFalpha, the anti-apoptotic activity of NF-kappa B involves suppression of the c-Jun N-terminal kinase (JNK) cascade. Using an unbiased screen, we have previously identified Gadd45 beta/Myd118, a member of the Gadd45 family of inducible factors, as a pivotal mediator of this suppressive activity of NF-kappa B. However, the mechanisms by which Gadd45 beta inhibits JNK signalling are not understood. Here, we identify MKK7/JNKK2--a specific and essential activator of JNK--as a target of Gadd45 beta, and in fact, of NF-kappa B itself. Gadd45 beta binds to MKK7 directly and blocks its catalytic activity, thereby providing a molecular link between the NF-kappa B and JNK pathways. Importantly, Gadd45 beta is required to antagonize TNFalpha-induced cytotoxicity, and peptides disrupting the Gadd45 beta/MKK7 interaction hinder the ability of Gadd45 beta, as well as of NF-kappa B, to suppress this cytotoxicity. These findings establish a basis for the NF-kappa B control of JNK activation and identify MKK7 as a potential target for anti-inflammatory and anti-cancer therapy. PMID: 14743220 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: 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] --------------------------------------------------------------- 16: J Mol Biol. 2003 Dec 12;334(5):1009-22. Structure of NFAT bound to DNA as a monomer. Stroud JC, Chen L. Department of Chemistry and Biochemistry, University of Colorado at Boulder, Campus Box 215, Boulder, CO 80309-0437, USA. The nuclear factor of activated T cells (NFAT) is a calcium-dependent transcription factor that cooperates with a myriad of partner transcription factors to regulate distinct transcription programs. Transcription activation by NFAT without the cooperation of co-stimulatory signals in lymphocytes can also impose a genetic program of anergy. Although the ternary NFAT1/Fos-Jun/DNA complex has been structurally characterized, how NFAT1 recognizes DNA in the absence of cooperative partners and how such a binary NFAT/DNA complex may lead to the assembly of distinct high-order NFAT transcription complexes are still poorly understood. We have determined the crystal structure of the entire Rel homology region (RHR) of human NFAT1 (NFATc2) bound to DNA as a monomer. We also present footprinting evidence that corroborates the protein-DNA contacts observed in the crystal structure. Our structural and biochemical studies reveal the mechanism by which the monomeric Rel protein NFAT recognizes its cognate DNA site. A remarkable feature of the binary NFAT/DNA complex is the conformational flexibility exhibited by NFAT1 in the four independent copies of the NFAT/DNA complex in the crystal structure, which may reflect a mechanism by which NFAT1 interacts with a variety of protein partners as it mediates disparate biological responses. PMID: 14643663 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Nat Struct Biol. 2003 Oct;10(10):800-6. Epub 2003 Aug 31. Structure of NFAT1 bound as a dimer to the HIV-1 LTR kappa B element. Giffin MJ, Stroud JC, Bates DL, von Koenig KD, Hardin J, Chen L. Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA. DNA binding by NFAT1 as a dimer has been implicated in the activation of host and viral genes. Here we report a crystal structure of NFAT1 bound cooperatively as a dimer to the highly conserved kappa B site from the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR). This structure reveals a new mode of dimerization and protein-DNA recognition by the Rel homology region (RHR) of NFAT1. The two NFAT1 monomers form a complete circle around the kappa B DNA through protein-protein interactions mediated by both their N- and C-terminal subdomains. The major dimer interface, formed by the C-terminal domain, is asymmetric and substantially different from the symmetric dimer interface seen in other Rel family proteins. Comparison to other NFAT structures, including NFAT5 and the NFAT1-Fos-Jun-ARRE2 complex, reveals that NFAT1 adopts different conformations and its protein surfaces mediate distinct protein-protein interactions in the context of different DNA sites. PMID: 12949493 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Mol Cell Biol. 2003 Apr;23(8):2749-61. NF-kappa B-dependent assembly of an enhanceosome-like complex on the promoter region of apoptosis inhibitor Bfl-1/A1. Edelstein LC, Lagos L, Simmons M, Tirumalai H, Gelinas C. Center for Advanced Biotechnology and Medicine and Graduate Program in Biotechnology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. Expression of the prosurvival Bcl-2 homologue Bfl-1/A1 is induced by NF-kappa B-activating stimuli, while B and T cells from c-rel knockout mice show an absolute defect in bfl-1/a1 gene activation. Here, we demonstrate NF-kappa B-dependent assembly of an enhanceosome-like complex on the promoter region of bfl-1. Binding of NF-kappa B subunit c-Rel to DNA nucleated the concerted binding of transcription factors AP-1 and C/EBP beta to the 5'-regulatory region of bfl-1. Optimal stability of the complex was dependent on proper orientation and phasing of the NF-kappa B site. Chromatin immunoprecipitation analyses demonstrated that T-cell activation triggers in vivo binding of endogenous c-Rel, c-Jun, C/EBP beta, and HMG-IC to the bfl-1 regulatory region, coincident with selective recruitment of coactivators TAFII250 and p300, SWI/SNF chromatin remodeling factor component BRG-1, and basal transcription factors TATA-binding protein (TBP) and TFIIB, as well as hyperacetylation of histones H3 and H4. These results highlight a critical role for NF-kappa B in bfl-1 transcription and point to the need for a complex and precise regulatory network to control bfl-1 expression. To our knowledge, this is the first demonstration of enhanceosome-mediated regulation of a cell death inhibitor. PMID: 12665576 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Toxicol Appl Pharmacol. 2003 Mar 15;187(3):147-61. Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen. Zhou HR, Islam Z, Pestka JJ. Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1224, USA. Bacterial lipopolysaccharide (LPS) elicits inflammation and endotoxic shock by inducing proinflammatory cytokine gene expression. The purpose of this study was to test the hypothesis that differential activation of transcription factor binding in the spleen correlates with proinflammatory cytokine gene expression in mice exposed to LPS. When proinflammatory cytokine expression in spleen was evaluated in mice injected ip with 4 mg/kg LPS over an 8-h period, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 mRNAs were elevated up to 5-, 6-, and 300-fold, respectively, over vehicle controls. Both TNF- alpha and IL-6 mRNA peaked at 2 h and begin to decline thereafter, whereas IL-1beta mRNA remained elevated from 2 to 8 h. The capacities of splenic nuclear proteins to bind to six different consensus transcriptional control motifs associated with proinflammatory cytokine promoters were also measured over 8 h. Electrophoretic mobility shift assay (EMSA) revealed that binding activity was markedly increased at 0.5 to 8 h for activator protein-1 (AP-1) as were CCAAT enhancer-binding protein (C/EBP) and nuclear factor kappaB (NF-kappaB) at 0.5 to 1.5 h. At 0.5 h, cyclic AMP response element (CRE)-binding protein (CREB) and binding was slightly elevated, whereas activator protein- 2 (AP-2) and specificity protein 1 (Sp1) binding were not affected. Antibody supershift EMSA and Western blot analysis confirmed that increased binding of these factors correlated with LPS-induced increases in nuclear concentrations of AP-1 (c-Jun, phosphorylated c-Jun, Jun D, and Jun B), C/EBPbeta, NF-kappaB (p50, p65, and c-Rel), CREB (CREB-1, CREB-2, and ATF-2), and AP-2alpha proteins. Remarkably, after 8 h, C/EBP, CREB, AP-2, and Sp1 binding activities were greatly depleted relative to both naive and corresponding vehicle controls. When mice were exposed to a second dose of LPS, 8 h after a 4 mg/kg priming dose, TNF-alpha and IL-6 mRNA responses were markedly impaired, suggesting that the mice were endotoxin tolerant at this time point. Taken together, the quiescent, active, and suppressive phases of transcription factor binding observed in this model were highly consistent with the rapid transient nature of LPS-induced proinflammatory cytokine expression in vivo as well as tolerance to secondary LPS exposure. PMID: 12662898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: J Biol Chem. 2003 Jan 17;278(3):1585-93. Epub 2002 Nov 7. A central role for the JNK pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factor-beta-driven SMAD3/4-specific gene expression. Verrecchia F, Tacheau C, Wagner EF, Mauviel A. INSERM U532, Institut de Recherche sur la Peau Hopital Saint-Louis, 75475 Paris Cedex 10, France. We have focused our attention on the molecular events underlying the antagonistic activities of pro-inflammatory cytokines against transforming growth factor-beta (TGF-beta)/SMAD signaling. Using jnk1/2-knockout (jnk(-/-)) and I kappa B kinase-gamma/nemo(-/-) fibroblasts, we have determined the specific roles played by the JNK/AP-1 and NF-kappa B/Rel pathways in this phenomenon. We demonstrate that, in a cellular context devoid of JNK activity (i.e. jnk(-/-) fibroblasts), interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) did not inhibit the formation of SMAD-DNA complexes and the resulting SMAD-driven transcription in response to TGF-beta. On the other hand, lack of NF-kappa B activity in nemo(-/-) fibroblasts did not affect the antagonistic effect of pro-inflammatory cytokines against TGF-beta. In the latter cell type, overexpression of antisense c-jun mRNA or of a dominant-negative form of MKK4 blocked the inhibitory activity of TNF-alpha, similar to what was observed in normal human dermal fibroblasts. Among JNK substrates, c-Jun and JunB (but not activating transcription factor-2) antagonized TGF-beta/SMAD signaling in a JNK-dependent manner. Overexpression of JNK1 in jnk(-/-) fibroblasts restored the ability of cytokines and Jun proteins to interfere with SMAD signaling. In junAA mouse embryo fibroblasts, in which c-Jun can no longer be phosphorylated by JNK, JunB substituted for c-Jun in mediating the cytokine effect against SMAD-driven transcription in a JNK-dependent manner. These results suggest a critical role for JNK-mediated c-Jun and JunB phosphorylation in transmitting the inhibitory effect of pro-inflammatory cytokines against TGF-beta-induced SMAD signaling. In addition, we demonstrate that such a JNK-dependent regulatory mechanism underlies the antagonistic activity of TNF-alpha against TGF-beta-induced up-regulation of type I and III collagens in fibroblasts. PMID: 12426318 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Int Immunopharmacol. 2002 Jul;2(8):1065-77. Lipopeptide adjuvants: monitoring and comparison of P3CSK4- and LPS-induced gene transcription. Muller MR, Wiesmuller KH, Jung G, Loop T, Humar M, Pfannes SD, Bessler WG, Mittenbuhler K. Institut fur Molekulare Medizin und Zellforschung der Universitat Freiburg, AK Tumorimmunologie/Vakzine, Germany. Bacteria-derived synthetic lipoproteins constitute potent macrophage activators in vivo and are effective stimuli, enhancing the immune response especially with respect to low or non-immunogenic compounds. N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3 -lysine (P3CSK4), exhibiting one of the most effective lipopeptide derivatives, represents a highly efficient immunoadjuvant in parenteral, oral, nasal and genetic immunization either in combination with or after covalent linkage to antigen. In order to further elucidate its molecular mode of action with respect to the transcriptional level, we focused our investigations on the P3CSK4-induced modulation of gene transcription. We could show that P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. P3CSK4 activates the expression of tumor suppressor protein p53 (p53), c-rel, inhibitor of nuclear factor kappa B (NFkappaB) alpha (IkappaB alpha), type 2 (inducible) nitric oxide (NO) synthase (iNOS), CD40-LR, intercellular adhesion molecule-1 (ICAM-1) and interleukin 1/6/15 (IL-1/6/15). We detected no activation of heat shock protein (HSP) 27, 60, 84 and 86, osmotic stress protein 94 (Osp 94), IL-12, extracellular signal-regulated protein kinase 1 (ERK1), p38 mitogen activated protein (MAP)-kinase (p38), c-Jun NH2-terminal kinase (JNK), signal transducer and activator of transcription 1 (STAT1), CD14 and caspase genes. Furthermore, we monitored inhibition of STAT6, Janus kinase 3 (Jak3) and cyclin D1/D3 gene transcription after stimulating bone marrow-derived macrophages (BMDM) with lipopeptide. In addition, we monitored significant differences after lipopeptide and lipopolysaccharide (LPS) stimulation of bone marrow-derived murine macrophages. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines. PMID: 12349944 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: 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] --------------------------------------------------------------- 23: J Toxicol Environ Health A. 2002 Aug 23;65(16):1161-80. Effects of vomitoxin (deoxynivalenol) on the binding of transcription factors AP-1, NF-kappaB, and NF-IL6 in raw 264.7 macrophage cells. Wong SS, Zhou HR, Pestka JJ. Department of Food Science and Human Nutrition and Institute for Environmental Toxicology, Michigan State University, East Lansing, MI 48824, USA. The effects of vomitoxin (VT) on the binding activity of three transcription factors critical to pro-inflammatory cytokine regulation were assessed in the RAW 264.7 murine macrophage model by electrophoretic mobility shift assay (EMSA). When cells were treated with 100 to 250 ng/ml of VT, activator protein-1 (AP-1 binding) was increased after 2 and 8 h. This effect was potentiated when cells were coincubated with lipopolysaccharide (LPS) (synchronous model) but not when preincubated with LPS (delayed synchronous model). Supershift EMSA revealed that VT preferentially induced JunB, JunD, phosphorylated c-Jun, c-Fos, and Fra-2 binding activities of the AP-1 family. Nuclear factor kappaB (NF-kappaB) binding was increased at 2 and 8 h in cells subjected to synchronous and delayed synchronous VT exposure in the presence of LPS. Supershift EMSA indicated that the p-50 and c-Rel subunits of NF-kappaB/ Rel were specifically affected. Nuclear factor-IL6 (NF-IL6) binding was increased at 2 and 8 h with or without LPS in synchronous and delayed synchronous VT-exposure models. Here, the C/EBPbeta subunit was primarily involved in enhanced NF-IL6 binding. The capacity of VT to elevate binding of AP-1, NF-kappaB, and NF-IL6 may contribute to the VT-mediated cytokine up-regulation in vitro and in vivo. The observations that VT was active in synchronous and delayed synchronous models suggest that macrophages activated simultaneously or prior to toxin exposure were vulnerable to the effects of this trichothecene. PMID: 12167214 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: 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] --------------------------------------------------------------- 25: Mol Cell. 2002 Apr;9(4):789-98. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Hu CD, Chinenov Y, Kerppola TK. Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Networks of protein interactions coordinate cellular functions. We describe a bimolecular fluorescence complementation (BiFC) assay for determination of the locations of protein interactions in living cells. This approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment. BiFC analysis was used to investigate interactions among bZIP and Rel family transcription factors. Regions outside the bZIP domains determined the locations of bZIP protein interactions. The subcellular sites of protein interactions were regulated by signaling. Cross-family interactions between bZIP and Rel proteins affected their subcellular localization and modulated transcription activation. These results attest to the general applicability of the BiFC assay for studies of protein interactions. PMID: 11983170 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: FASEB J. 2002 Apr;16(6):529-38. Activation of an alternative NF-kappaB pathway in skeletal muscle during disuse atrophy. Hunter RB, Stevenson E, Koncarevic A, Mitchell-Felton H, Essig DA, Kandarian SC. Boston University, Department of Health Sciences, Boston, Massachusetts 02215, USA. Although cytokine-induced nuclear factor kappaB (NF-kappaB) pathways are involved in muscle wasting subsequent to disease, their potential role in disuse muscle atrophy has not been characterized. Seven days of hind limb unloading led to a 10-fold activation of an NF-kappaB-dependent reporter in rat soleus muscle but not the atrophy-resistant extensor digitorum longus muscle. Nuclear levels of p50 were markedly up-regulated, c-Rel was moderately up-regulated, Rel B was down-regulated, and p52 and p65 were unchanged in unloaded solei. The nuclear IkappaB protein Bcl-3 was increased. There was increased binding to an NF-kappaB consensus oligonucleotide, and this complex bound antibodies to p50, c-Rel, and Bcl-3 but not other NF-kappaB family members. Tumor necrosis factor alpha (TNF-alpha) and TNF receptor-associated factor 2 protein were moderately down-regulated. There was no difference in p38, c-Jun NH(2)-terminal kinase or Akt activity, nor were activator protein 1 or nuclear factor of activated T cell-dependent reporters activated. Thus, whereas several NF-kappaB family members are up-regulated, the prototypical markers of cytokine-induced activation of NF-kappaB seen with disease-related wasting are not evident during disuse atrophy. Levels of an anti-apoptotic NF-kappaB target, Bcl-2, were increased fourfold whereas proapoptotic proteins Bax and Bak decreased. The evidence presented here suggests that disuse muscle atrophy is associated with activation of an alternative NF-kappaB pathway that involves the activation of p50 but not p65. PMID: 11919155 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Biochem J. 2002 Apr 1;363(Pt 1):175-82. Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells. Tao L, Wadsworth S, Mercer J, Mueller C, Lynn K, Siekierka J, August A. R. W. Johnson Pharmaceutical Research Institute, Drug Discovery Research, Raritan, NJ 08869, USA. T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-kappa B inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element. PMID: 11903060 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Nature. 2001 Nov 15;414(6861):308-13. Comment in: Nature. 2001 Nov 15;414(6861):265-6. Nature. 2003 Aug 14;424(6950):741; discussion 742. Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. De Smaele E, Zazzeroni F, Papa S, Nguyen DU, Jin R, Jones J, Cong R, Franzoso G. The Gwen Knapp Center for Lupus and Immunology research, The University of Chicago, Illinois 60637, USA. In addition to coordinating immune and inflammatory responses, NF-kappaB/Rel transcription factors control cell survival. Normally, NF-kappaB dimers are sequestered in the cytoplasm by binding to inhibitory IkappaB proteins, and can be activated rapidly by signals that induce the sequential phosphorylation and proteolysis of IkappaBs. Activation of NF-kappaB antagonizes apoptosis or programmed cell death by numerous triggers, including the ligand engagement of 'death receptors' such as tumour-necrosis factor (TNF) receptor. The anti-apoptotic activity of NF-kappaB is also crucial to oncogenesis and to chemo- and radio-resistance in cancer. Cytoprotection by NF-kappaB involves the activation of pro-survival genes; however, its basis remains poorly understood. Here we report that NF-kappaB complexes downregulate the c-Jun amino-terminal kinase (JNK) cascade, thus establishing a link between the NF-kappaB and the JNK pathways. This link involves the transcriptional upregulation of gadd45beta/myd118 (ref. 4), which downregulates JNK signalling induced by the TNF receptor (TNF-R). This NF-kappaB-dependent inhibition of the JNK pathway is central to the control of cell death. Our findings define a protective mechanism that is mediated by NF-kappaB complexes and establish a role for the persistent activation of JNK in the apoptotic response to TNF-alpha. PMID: 11713530 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Cell Neurosci. 2001 Sep;18(3):320-31. Analysis of the NF-kappa B and PI 3-kinase/Akt survival pathways in nerve growth factor-dependent neurons. Sarmiere PD, Freeman RS. Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, New York 14642, USA. Nerve growth factor (NGF) readdition to NGF-deprived neurons can halt Jun N-terminal kinase (JNK) activation, cytochrome c release, and cell death through mechanisms that may involve phosphatidylinositol (PI) 3-kinase, Akt, and nuclear factor kappa B (NF-kappaB). We found that expression of the NF-kappaB protein c-Rel in NGF-deprived neurons blocks cytochrome c release but does not inhibit c-Jun phosphorylation. Conversely, inhibition of NF-kappaB in NGF-maintained neurons promotes cytochrome c release and cell death. In contrast to c-Rel, activated PI 3-kinase and Akt inhibit c-Jun phosphorylation but have only a small effect on cytochrome c release. Finally, although c-Rel can protect neurons from death caused by inhibitors of PI 3-kinase or Akt, NF-kappaB function is not critical for Akt-promoted survival. These results suggest that the PI 3-kinase/Akt and NF-kappaB survival pathways target distinct cell death events in neurons. Copyright 2001 Academic Press. PMID: 11591132 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Int Immunopharmacol. 2001 Jul;1(7):1331-9. Experimental evidences and signal transduction pathways involved in the activation of NF-kappa B/Rel by angelan in murine macrophages. Jeon YJ, Kim HM. Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305-600, South Korea. In our previous studies, we showed that angelan, a polysaccharide purified from Angelica gigas Nakai, activated macrophages to induce the translocation of NF-kappa B/Rel into nucleus and DNA binding to its cognate site in the promoter of iNOS gene [Immunopharmacology 43 (1999) 1; Immunopharmacology 49 (2000) 275]. In the present study, we showed that angelan induces the transcriptional activation of NF-kappa B/Rel and investigated the intracellular signal transduction pathways involved in the angelan-induced NF-kappa B/Rel activation by murine macrophages. Treatment of RAW 264.7 cells with angelan resulted in significant activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38, while stress-activated protein kinase/c-Jun NH2 terminal kinase (SAPK/JNK) was not activated by angelan. The specific p38 inhibitor SB203580 abrogated the angelan-induced NF-kappa B/Rel activation, whereas the selective MAPK/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD98059 did not affect the NF-kappa B/Rel induction. Treatment of RAW 264.7 cells with both anti-CD14 Ab and anti-CR3 Ab significantly blocked angelan-induced NF-kappa B/Rel activation. In conclusion, we demonstrate that angelan induces NF-kappa B/Rel activation through the CD14 and CR3 membrane receptor and p38 kinase that is critically involved in the signal transduction leading to NF-kappa B/Rel activation in murine macrophages. PMID: 11460313 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: J Immunol. 2001 Apr 1;166(7):4560-9. HIV-1 Tat inhibits IL-2 gene transcription through qualitative and quantitative alterations of the cooperative Rel/AP1 complex bound to the CD28RE/AP1 composite element of the IL-2 promoter. Gonzalez E, Punzon C, Gonzalez M, Fresno M. Centro de Biologia Molecular Severo Ochoa Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Cantoblanco, Madrid, Spain. Dysregulation of cytokine secretion plays an important role in AIDS pathogenesis. Here, we demonstrate that expression of HIV-1 Tat protein in Jurkat cells induces a severe impairment of IL-2 but not TNF gene transcription. Interestingly, this inhibition correlates with the effect of the viral protein on the transactivation of the CD28RE/AP1 composite element (-164/-154), but not with that observed on the NFAT/AP1 site of the IL-2 gene promoter, neither with the effect on NF-kappa B- nor AP1-independent binding sites. Endogenous expression of Tat induced a decrease in the amount of the specific protein complex bound to the CD28RE/AP1 probe after PMA plus calcium ionophore stimulation. This effect was accompanied by qualitative alterations of the AP1 complex. Thus, in wild-type Jurkat cells, c-jun was absent from the complex, whereas in Tat-expressing cells, c-jun was increasingly recruited overtime. By contrast, similar amounts of c-rel and a small amount of NFAT1 were detected both in wild type and in Jurkat Tat(+) cells. Furthermore, Tat not only induced the participation of c-jun in the cooperative complex but also a decrease in its transactivation activity alone or in combination with c-rel. Thus, the interaction of Tat with the components of this rel/AP1 cooperative complex seems to induce quantitative and qualitative alterations of this complex as activation progresses, resulting in a decrease of IL-2 gene transcription. Altogether our results suggest the existence of tuned mechanisms that allow the viral protein to specifically affect cooperative interactions between transcription factors. PMID: 11254713 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Immunol. 2001 Feb 15;166(4):2437-43. Identification of a CD28 response element in the CD40 ligand promoter. Parra E, Mustelin T, Dohlsten M, Mercola D. Sidney Kimmel Cancer Center, San Diego, CA 92121, USA. eparra@skcc.org Ligation of the T cell coreceptor CD28 or CD2 by its cognate ligands B7-1 or LFA-3, respectively, greatly aids the Ag-induced up-regulation of several genes, including IL-2 and CD40 ligand (CD40L). Using luciferase reporter constructs under the control of the 1.2 kb of 5' noncoding region of the human CD40L gene, we have found that stimulation through CD28 was required for a strong transcriptional activity of the CD40L promoter in response to TCR ligation, while the activity induced by CD2 was slightly lower than CD28. Deletion analysis demonstrated that the transcriptional elements mediating this effect were located within a 300-bp region upstream of the start site. Further dissection of this region and gel shift analyses demonstrated the presence of a CD28 response element in a region located between nucleotides -170 to -164 relative to the start site. Transcriptional studies with a CD40L enhancer-promoter carrying a mutation in this putative CD28 response element revealed that the activity was reduced by 80 and 70% after B7-1 and LFA-3 costimulation, respectively. The transcription factor complex bound to this site contained at least JunD, c-Fos, p50, p65, and c-REL:, but not c-Jun. Mutations introduced into the CD28RE also blocked the binding of this complex. These observations identify an important role for the CD28 signaling pathway in the regulation of CD40L promoter transcriptional activity. PMID: 11160303 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Thromb Haemost. 2000 Oct;84(4):712-21. Verotoxin-1 induces tissue factor expression in human umbilical vein endothelial cells through activation of NF-kappaB/Rel and AP-1. Ishii H, Takada K, Higuchi T, Sugiyama J. Department of Public Health, Showa Pharmaceutical University, Higashi Tamagawa Gakuen, Machida, Tokyo, Japan. h-ishii@ac.shoyaku.ac.jp This study examined the effect of verotoxin-1 (VT-1), which is released from Escherichia coli O157:H7, on endothelial expression of tissue factor (TF), a cofactor required to initiate blood coagulation. In order to elucidate the molecular basis for development of hemolytic uremic syndrome (HUS) in patients infected with E. coli O157:H7, human umbilical vein endothelial cells (HUVECs) were exposed to purified VT-1. VT-1 increased both TF activity and TF mRNA in HUVECs without loss of cell viability in a time- and dose-dependent manner from 0.1 to 10 ng/ml VT-1. Nuclear proteins extracted from VT-1-stimulated HUVECs bound to the consensus NF-kappaB/Rel and AP-1 binding oligonucleotides in a dose-dependent manner within 2 h after the stimulation in electrophoretic mobility shift assays (EMSA). Nuclear proteins from VT-1-stimulated HUVECs formed two complexes with the NF-kappaB/Rel binding motif in the human TF promoter (TF-kappaB motif). The supershift assays, using antibodies for human p65, p50 or c-Rel, indicated that the lower complex was composed of p65/p50 and the higher complex was a p65 homo- or hetero-dimer with the Rel family, except c-Rel. The human TF promoter contains two AP-1 binding sites, the proximal and distal AP-1 binding sites. The supershift assays indicated that AP-1 containing mainly c-Jun and JunD, positively bound to the proximal AP-1 motif of TF (TF-AP-1). The distal TF-AP-1 motif did not show positive binding with nuclear proteins from VT-1-stimulated HUVECs. Pretreatment of HUVECs with curcumin, an inhibitor of NF-kappaB/Rel activation, synthesis of c-Jun mRNA and binding of activated AP- I with AP-binding oligonucleotide, prevented the VT-1 induced increase in TF mRNA and activity in VT-1-stimulated HUVECs. Curcumin also inhibited NF-kappaB and AP-1 binding to TF-kappaB and proximal TF-AP-1 oligonucleotides, respectively, in a dose-dependent manner. The present work suggests that both the NF-kappaB/Rel and AP-1 activated in endothelial cells by stimulation with VT-1 binds to the TF-kappaB and proximal AP-1 binding sites, respectively, of the TF promoter. PMID: 11057875 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Gastroenterology. 2000 Nov;119(5):1209-18. Suppression of NF-kappaB activity by sulfasalazine is mediated by direct inhibition of IkappaB kinases alpha and beta. Weber CK, Liptay S, Wirth T, Adler G, Schmid RM. Department of Internal Medicine I, University of Ulm, Ulm, Germany. BACKGROUND & AIMS: Activation of NF-kappaB/Rel has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Various drugs used in the treatment of IBD, such as glucocorticoids, 5-aminosalicylic acid, and sulfasalazine, interfere with NF-kappaB/Rel signaling. The aim of this study was to define the molecular mechanism by which sulfasalazine inhibits NF-kappaB activation. METHODS: The effects of sulfasalazine and its moieties on NF-kappaB signaling were evaluated using electromobility shift, transfection, and immune complex kinase assays. The direct effect of sulfasalazine on IkappaB kinase (IKK) activity was investigated using purified recombinant IKK-alpha and -beta proteins. RESULTS: NF-kappaB/Rel activity induced by tumor necrosis factor alpha, 12-O-tetradecanoylphorbol-13-acetate, or overexpression of NF-kappaB-inducing kinase, IKK-alpha, IKK-beta, or constitutively active IKK-alpha and IKK-beta mutants was inhibited dose dependently by sulfasalazine. Sulfasalazine inhibited tumor necrosis factor alpha-induced activation of endogenous IKK in Jurkat T cells and SW620 colon cells, as well as the catalytic activity of purified IKK-alpha and IKK-beta in vitro. In contrast, the moieties of sulfasalazine, 5-aminosalicylic acid, and sulfapyridine or 4-aminosalicylic acid had no effect. Activation of extracellular signal-related kinase (ERK) 1 and 2, c-Jun-N-terminal kinase (JNK) 1, and p38 was unaffected by sulfasalazine. The decrease in substrate phosphorylation by IKK-alpha and -beta is associated with a decrease in autophosphorylation of IKKs and can be antagonized by excess adenosine triphosphate. CONCLUSIONS: These data identify sulfasalazine as a direct inhibitor of IKK-alpha and -beta by antagonizing adenosine triphosphate binding. The suppression of NF-kappaB activation by inhibition of the IKKs contributes to the well-known anti-inflammatory and immunosuppressive effects of sulfasalazine. PMID: 11054378 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Immunopharmacology. 2000 Jul 20;48(2):173-83. Dexamethasone inhibits IL-1 beta gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-kappa B/Rel and AP-1 activation. Jeon YJ, Han SH, Lee YW, Lee M, Yang KH, Kim HM. Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, 305-600, Taejon, South Korea. yjjeon@bioneer.kaist.ac.kr In the present study, the mechanism by which dexamethasone (DEX) inhibited IL-1beta gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in LPS-induced IL-1beta mRNA expression was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Since the promoter in IL-1beta gene contains binding motifs for NF-kappaB/Rel, AP-1, NF-IL6, and CREB/ATF, which appear to be important in LPS-mediated IL-1beta induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-kappaB/Rel and AP-1 in chloramphenicol acetyltransferase activity, while neither NF-IL6 nor CREB/ATF activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-kappaB/Rel and AP-1 proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay (EMSA). DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. DEX treatment of RAW 264.7 cells did not inhibit the nuclear translocation of c-jun and c-fos. We found that the inhibition of IL-1beta production by DEX is not related to p38, which is important in the IL-1beta induction. These results suggest that DEX may inhibit IL-1beta gene expression by a mechanism involving the blocking of LPS-induced NF-kappaB/Rel and AP-1 activation. PMID: 10936515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Cell Immunol. 2000 May 1;201(2):109-23. Differential regulation of CD40-mediated human B cell responses by antibodies directed against different CD40 epitopes. Sakata N, Hamelmann E, Siadak AW, Terada N, Gerwins P, Aruffo A, Johnson GL, Gelfand EW. Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO 80206, USA. Ligation of CD40 using anti-CD40 or soluble CD40-ligand activates numerous intracellular kinases which transduce signals to the nucleus. The nature whereby these signaling events are coupled to distal functional events in B cells is poorly understood. In this study, using anti-CD40 monoclonal antibodies which recognize different epitopes on CD40, we compare the ability to activate the stress-activated protein kinases (SAPK) such as c-Jun NH(2) terminal kinase and p38 in human B cells with CD40 function. Activation of the SAPK pathway correlated with levels of activation of Rel/NF-kappaB transcription factors, but did not appear to be associated with rescue from anti-IgM induced apoptosis by suppressing caspase (CPP32) activity. Somewhat surprisingly, in the presence of IL-4, those antibodies to CD40 which failed to activate SAPK were most active in IgE production. IgE production was augmented in the presence of wortmannin. These studies suggest that rescue from apoptosis and IgE production mediated via CD40 may be independent of SAPK activation, induction of Rel/NF-kappaB, or suppression of CPP32 and that IgE production is, at least in part, regulated by signaling pathways that are dependent on phosphatidylinositol 3-kinase. Copyright 2000 Academic Press. PMID: 10831320 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: 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] --------------------------------------------------------------- 38: J Biol Chem. 2000 Jun 16;275(24):18432-40. Glucocorticoids suppress tumor necrosis factor-alpha expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B and c-Jun-activating transcription factor-2 binding sites in the promoter. Steer JH, Kroeger KM, Abraham LJ, Joyce DA. Department of Pharmacology, University of Western Australia, Nedlands, Western Australia, Australia 6907. Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter. PMID: 10748079 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: J Neurochem. 1999 Dec;73(6):2501-9. Activation of AP-1 and nuclear factor-kappaB transcription factors is involved in hydrogen peroxide-induced apoptotic cell death of oligodendrocytes. Vollgraf U, Wegner M, Richter-Landsberg C. University of Oldenburg, Department of Biology, Germany. H2O2-induced onset and execution of programmed cell death in mature rat brain oligodendrocytes in culture is accompanied by the induction and nuclear translocation of the transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB), both of which have been discussed as regulators of cell death and survival. Supershift analysis of nuclear extracts indicated that the AP-1 complex consists of c-Jun, c-Fos, JunD, and possibly JunB proteins, and that the NF-kappaB complex contains p50, p65, and c-Rel proteins. The first signs of DNA fragmentation were seen already during the first hour after the treatment. DNA fragmentation could be prevented by the antioxidants pyrrolidine dithiocarbamate and vitamin E, by the nuclease inhibitor aurintricarboxylic acid, and by preincubation with the iron chelator deferoxamine (DFO). Additionally, DFO protected oligodendrocytes from H2O2-induced cytotoxic effects as assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and suppressed the formation of free radicals. DFO alone led to a slight increase and in combination with H2O2 synergistically induced DNA-binding activities of AP-1 and NF-kappaB in oligodendrocytes. Our data suggest that although low levels of H2O2 directly activate AP-1 and NF-kappaB and might contribute to signal transduction pathways promoting cell survival, the formation and action of hydroxyl radicals promote cell death mechanisms that can be attenuated by the iron chelator DFO. PMID: 10582611 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: 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] --------------------------------------------------------------- 41: Mol Carcinog. 1999 Oct;26(2):119-29. Constitutive activation of transcription factors NF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines. Ondrey FG, Dong G, Sunwoo J, Chen Z, Wolf JS, Crowl-Bancroft CV, Mukaida N, Van Waes C. Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA. We previously reported that human head and neck squamous cell carcinomas (HNSCCs) express the pro-inflammatory and pro-angiogenic cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in vitro and in vivo. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear factor (NF) kappaB/Rel A, activator protein-1 (AP-1), and CCAAT enhancer-binding protein beta (C/EBPbeta, or NF-IL6), which have been reported to contribute to activation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro-inflammatory cytokines, by using electrophoretic mobility shift and reporter-gene assays. Constitutive activation of NF-kappaB, AP-1, and NF-IL6 DNA-binding proteins was detected. Supershift analysis with antibodies specific for NF-kappaB, AP-1, and NF-IL6 binding proteins showed that the NF-kappaB-binding protein included p65/Rel A and p50; AP-1 activity included c-jun, junB, junD, and Fra-1; and NF-IL6 included C/EBPbeta. Mutational analysis of the NF-kappaB, AP-1, and NF-IL6 sites in the IL-8 promoter region showed that NF-kappaB and AP-1 sites contributed to constitutive IL-8 reporter activity in HNSCC. HNSCC lines that exhibited increased IL-8 secretion relative to simian virus 40-immortalized and primary keratinocyte cell lines also demonstrated a concordant increase in NF-kappaB reporter activity relative to nonmalignant keratinocytes. We concluded that the early transcription factors NF-kappaB, AP-1, and NF-IL6 are constitutively activated in human HNSCC cell lines and that NF-kappaB and AP-1 promote expression of the pro-inflammatory and pro-angiogenic cytokine IL-8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and other early gene products that contribute to pathogenesis of the malignant phenotype in HNSCC and in defining potential targets for pharmacologic and molecular therapy of HNSCC. Mol. Carcinog. 26:119-129, 1999. Published 1999 Wiley-Liss, Inc. PMID: 10506755 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: J Cell Physiol. 1999 Nov;181(2):361-70. Differential expression and regulation of chemokines JE, KC, and IP-10 gene in primary cultured murine hepatocytes. Wang H, Gao X, Fukumoto S, Tademoto S, Sato K, Hirai K. Department of Medical Zoology, Faculty of Medicine, Tottori University, Yonago, Japan. Chemokines are a superfamily of structurally related chemoattractant cytokines. JE (monocyte chemoattractant protein-1) and IP-10 (interferon-inducible protein-10) have been detected in the diseased liver. However the in vitro expression is unclear. In this report, we revealed that JE, KC (melanoma growth-stimulating activity gene), and IP-10 mRNAs are not expressed in the normal liver but spontaneously and time-dependently expressed in the primary hepatocytes. The serum-independent gene expression of both JE and KC lasted over 72 h, but that of IP-10 became undetectable 24 h after isolation with collagenase perfusion method. The induction of the genes' expression was not due to LPS contamination but nevertheless was associated with isolation procedure. Actinomycin D blocked their expression. The increase of their transcripts resulted from greater increase in gene transcription and lower mRNA stability. Consistent with c-jun, their mRNA expressions were simultaneously superinduced by cycloheximide (1 microg/ml), suggesting that de novo protein synthesis is involved their transcriptions. Inhibition by pyrrolidine dithiocarbamate (PDTC), a NF-kappaB/c-rel inhibitor, and EMSA imply that NF-kappaB/c-rel is important in their expressions. Of particular interest is that dexamethasone upregulated the spontaneous expression of KC, but suppressed that of JE and IP-10. LPS upregulated the mRNA levels of JE and KC but did not affect that of IP-10. IFN-gamma induced the expression of IP-10; however unlike in macrophages, it did not selectively inhibit that of JE and KC. Our data demonstrated the existence and differential gene expression of JE, KC, and IP-10 in primary cultured hepatocytes, and these are considered to be a reflex of the alteration of hepatocyte cellular physiology during and after isolation. Copyright 1999 Wiley-Liss, Inc. PMID: 10497315 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: 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] --------------------------------------------------------------- 44: Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7214-9. NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Lopez-Rodriguez C, Aramburu J, Rakeman AS, Rao A. Department of Pathology, Harvard Medical School, The Center for Blood Research, Boston, MA 02115, USA. NFAT transcription factors are related to NF-kappaB/Rel proteins and form cooperative complexes with Fos and Jun on DNA. We have identified an NFAT-related protein, NFAT5, which differs from the conventional NFAT proteins NFAT1-4 in its structure, DNA binding, and regulation. NFAT5 contains a NFAT-like Rel homology domain, conserves the DNA contact residues of NFAT1-4, and binds DNA sequences similar to those found in the regulatory regions of well-characterized NFAT-dependent genes. However, it lacks the majority of Fos/Jun contact residues and does not bind cooperatively with Fos and Jun to DNA. Unlike NFAT1-4, whose nuclear import is tightly regulated by calcineurin-mediated dephosphorylation, NFAT5 is a constitutively nuclear phosphoprotein regardless of calcineurin activation. These features suggest that unlike the conventional NFAT proteins, NFAT1-4, which activate gene transcription by integrating inputs from calcium/calcineurin and protein kinase C/mitogen-activated protein kinase signaling pathways, NFAT5 participates in as-yet-unidentified signaling pathways in diverse immune and nonimmune cells. PMID: 10377394 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Oncogene. 1999 Jun 3;18(22):3316-23. The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators. Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau S, Wulczyn FG, Scheidereit C, Leutz A. Max-Delbruck-Center for Molecular Medicine MDC, Berlin. The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase. PMID: 10362352 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Clin Cancer Res. 1999 May;5(5):1197-202. Activation of nuclear factor-kappa B in human metastatic melanomacells and the effect of oxidative stress. Meyskens FL Jr, Buckmeier JA, McNulty SE, Tohidian NB. Department of Medicine and the Chao Family Comprehensive Cancer Center, University of California-Irvine, Orange 92868, USA. flmeyske@uci.edu The biological basis for the general pharmacological resistance of human melanoma is unknown. A unique biochemical feature of the melanocyte is the synthesis of melanin, which leads to the generation of hydrogen peroxide and the consumption of reduced glutathione. This activity produces a state of chronic oxidative stress in these cells. We demonstrated previously that the expression of the c-jun family was dysregulated in metastatic melanoma cells compared with normal human melanocytes (D. T. Yamanishi et al., J. Invest. Dermatol., 97: 349-353, 1991). In the current investigation, we measured the levels of two major redox response transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1, in metastatic melanoma cells and normal melanocytes and their response to oxidative stress. The basal DNA-binding activity of NF-kappaB as measured by the electrophoretic mobility shift assay in metastatic melanoma cells was increased 4-fold compared with that of normal melanocytes. This level of binding was paralleled by a 1.5- to 4-fold increase in the expression of p50 (NF-kappaB1), p65 (Rel-A), and IkappaB-alpha as measured by Northern blot analysis. In contrast, the expression of p75 (c-rel) was markedly decreased (60%) in melanoma cells compared with normal melanocytes. Following oxidative stress produced by enzyme-generated H2O2, free H2O2, or incubation with buthionine sulfoximine, NF-kappaB binding activity increased 1.5- to 2.5-fold in melanoma cells (buthionine sulfoximine > H2O2), but only slightly in normal melanocytes. In contrast, activator protein-1 binding activity was unaffected or increased in normal melanocytes in response to oxidative stress, but was either unaffected or decreased in melanoma cells. These results suggest that the redox regulation of melanoma cells at the molecular level is fundamentally different from normal melanocytes and may offer a unique avenue for preventive or therapeutic intervention as well as new insights into the pathogenesis of melanocyte transformation. PMID: 10353757 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: J Immunol. 1999 May 15;162(10):5805-12. Costimulation reverses the defect in IL-2 but not effector cytokine production by T cells with impaired IkappaBalpha degradation. Aune TM, Mora AL, Kim S, Boothby M, Lichtman AH. Departments ofMedicine (Rheumatology) and Microbiology/Immunology, Vanderbilt University Medical School, Nashville, TN 37232, USA. Although the transcriptional basis for states of unresponsiveness in primary T cells is unclear, tolerant B lymphocytes exhibit inhibition of both c-Jun N-terminal kinase induction and IkappaBalpha (inhibitor of NF-kappaBalpha) degradation, leading to lower levels of both nuclear AP-1 and NF-kappaB. Expression of an IkappaBalpha mutant resistant to signal-induced degradation in transgenic T cells caused markedly deficient effector cytokine (IL-4, IFN-gamma) production after primary TCR stimulation despite a detectable level of nuclear NF-kappaB. A TCR response element from the IFN-gamma promoter, despite lacking detectable NF-kappaB/Rel sites, was also unresponsive to TCR ligation. Nuclear induction of AP-1 proteins in response to T cell activation was diminished in transgenic T cells. Costimulation induced by anti-CD28 mAb increased IL-2 production, but failed to reverse the defects in effector cytokine production. Taken together, these data indicate that impaired NF-kappaB/Rel signaling in T cells interferes with the signal transduction pathways required for efficient induction of effector cytokine production. PMID: 10229814 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: J Immunol. 1999 Mar 15;162(6):3176-87. The Jun kinase cascade is responsible for activating the CD28 response element of the IL-2 promoter: proof of cross-talk with the I kappa B kinase cascade. Kempiak SJ, Hiura TS, Nel AE. Department of Medicine, University of California, School of Medicine, Los Angeles 90095, USA. Costimulation of TCR/CD3 and CD28 receptors leads to activation of the Jun kinase (JNK) cascade, which plays a key role in T cell activation, including activation of the IL-2 promoter. We demonstrate that the JNK cascade plays a central role in the activation of the CD28 response element (CD28RE) in the IL-2 promoter. This response element is linked to an activating protein-1 (AP-1) site, which functions synergistically with the CD28RE. The role of the JNK cascade in the activation of this composite element is twofold: 1) activation of the AP-1 site through transcriptional activation of c-Jun, and 2) activation of the CD28RE through selective cross-talk with I kappa B kinase-beta (IKK beta). Dominant-negative versions of JNK kinase, c-Jun, and IKK beta interfered In CD3- plus CD28-induced CD28RE/AP-1 luciferase activity in Jurkat cells. In contrast, the dominant-active JNK kinase kinase, MEKK1, induced CD28RE/AP-1 luciferase activity, in parallel with induction of c-Jun and c-Rel binding to this combined promoter site. Dominant-active MEKK1 also induced transfected IKK beta, but not IKK alpha, activity. In contrast to the JNK cascade, the extracellular signal-regulated kinase (ERK) cascade did not exert an affect on the CD28RE/AP-1 site, but did contribute to activation of the distal NF-AT/AP-1 site. PMID: 10092768 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Cytokine. 1999 Jan;11(1):16-28. Involvement of NF-kappaB p50/p65 heterodimer in activation of the human pro-interleukin-1beta gene at two subregions of the upstream enhancer element. Goto M, Katayama KI, Shirakawa F, Tanaka I. Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan. m4-gotou@eisai.co.jp A region between-3134 and -2729 bp upstream from the transcription site of the human pro-interleukin 1beta (proIL-1beta) gene was identified as an LPS-responsive enhancer element. In this study, the influence of the sequences located between -3134 and -2987 on the transcriptional activity of the proIL-1beta gene in LPS-stimulated Raw 264.7 cells was examined in detail. The results obtained by transient transfection of fos -CAT constructs that contained serial 5'-deletion mutations showed that the region between -3134 and -3059 appears to be required for the induction of transcription by LPS. Gel shift assay studies with synthetic oligonucleotides corresponding to partial sequences of the latter region and nuclear extracts from stimulated cells revealed specific protein binding sites between -3110 and -3090 and between -3079 and -3059. These specific bindings were time and LPS dose dependent. The results of supershift analysis using specific antibodies against transcription factors suggested that both binding complexes contained the NF-kappaB components p50 and p65, and did not contain other NF-kappaB proteins (p52, c-Rel, Rel B), AP-1 proteins (c-Fos, C-Jun), CREB or C/EBPbeta (NF-IL6). Mutation of either of the putative NF-kappaB-binding sites in the enhancer element decreased the LPS-stimulated transcriptional activity. These data indicated that two NF-kappaB-binding sites, which are located between -3134 and -3059, are critical for the activation of proIL-1beta gene transcription. Copyright 1999 Academic Press. PMID: 10080875 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Toxicol Appl Pharmacol. 1999 Mar 15;155(3):280-6. Lead activates nuclear transcription factor-kappaB, activator protein-1, and amino-terminal c-Jun kinase in pheochromocytoma cells. Ramesh GT, Manna SK, Aggarwal BB, Jadhav AL. College of Pharmacy and Health Sciences, Texas Southern University, Houston, Texas, 77004, USA. Lead (Pb) is a ubiquitous environmental contaminant that produces variety of effects on the central and peripheral nervous system, induces inflammatory response, and modulates immune functions. Though increase in lipid peroxidation and reactive oxygen intermediates (ROI) have been observed in Pb-induced toxicity, the molecular mechanism underlying these effects is largely unknown. Since nuclear factor kappa B (NF-kappaB) and activator protein (AP-1) are known to be activated by oxidative stress, we hypothesized that Pb-induced effects may be modulated via these transcription factors. The effects of Pb on NF-kappaB, AP-1, and related kinases were studied in pheochromocytoma cells (PC-12). Our results showed that treatment of murine PC-12 cells with Pb resulted in activation of NF-kappaB and degradation of IkappaBalpha (the inhibitory subunit of NF-kappaB). Pb-induced NF-kappaB dependent gene expression was also enhanced. The binding of Pb-induced NF-kappaB to DNA was blocked by antibodies for p65 and p50 but not by c-Rel or nonspecific antibodies such as cyclin D-1 and preimmune serum, suggesting that NF-kappaB consisted of p65 and p50 subunits. Similar to its effects on NF-kappaB, Pb also activated AP-1 in a time- and dose-dependent manner. Besides activating these transcription factors, Pb was also found to upregulate the related kinases such as mitogen activated protein kinase kinase (MEK) and c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) in a dose- and time-dependent manner. Thus, these results suggest that NF-kappaB, AP-1, MEK, and JNK may be important mediators of Pb-induced signaling in gene expression mediating inflammatory response and immunomodulation. Copyright 1999 Academic Press. PMID: 10079214 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Arterioscler Thromb Vasc Biol. 1999 Feb;19(2):419-26. Resveratrol, a polyphenolic compound found in wine, inhibits tissue factor expression in vascular cells : A possible mechanism for the cardiovascular benefits associated with moderate consumption of wine. Pendurthi UR, Williams JT, Rao LV. Departments of Molecular Biology and Biochemistry, The University of Texas Health Center at Tyler, 75708, USA. usha@uthct.edu A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease mortality. The phytochemical resveratrol found in wine, derived from grapes, has been thought to be responsible for cardiovascular benefits associated with wine consumption because it was shown to have antioxidant and antiplatelet activities. In the present investigation, we examined the effect of resveratrol on induction of tissue factor (TF) expression in vascular cells that were exposed to pathophysiological stimuli. The data presented herein show that resveratrol, in a dose-dependent manner, inhibited the expression of TF in endothelial cells stimulated with a variety of agonists, including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS). A similar inhibition of TF induction was also seen in LPS stimulated monocytes that were pretreated with resveratrol before their stimulation with LPS. In addition, resveratrol was shown to inhibit the LPS-induced expression of TNFalpha mRNA in endothelial cells and of TNFalpha and IL-1beta mRNA in monocytes. Nuclear run-on analysis in endothelial cells showed that resveratrol inhibited TF expression at the level of transcription. However, resveratrol did not significantly alter the binding of the transcription factors c-Fos/c-Jun and c-Rel/p65, the transcription factors required for the induction of TF promoter in both endothelial cells and monocytes. Similarly, resveratrol had no significant effect on the binding of NF-kappaB in endothelial cells stimulated with IL-1beta, TNFalpha, and LPS. Overall, our data show that resveratrol could effectively suppress the aberrant expression of TF and cytokines in vascular cells, but it requires further investigation to understand how resveratrol exerts its inhibitory effect. PMID: 9974427 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: J Biol Chem. 1999 Jan 8;274(2):987-92. Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor kappaB. Kasibhatla S, Genestier L, Green DR. Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA. skasibhatla@liai.org T cell receptor engagement activates transcription factors important for cytokine gene regulation. Additionally, this signaling pathway also leads to activation-induced apoptosis in T lymphocytes that is dependent on FasL transcription and expression. Here we demonstrate that nuclear factor kappaB (NF-kappaB), which is involved in the transcriptional regulation of many cytokine genes expressed in activated lymphocytes, also plays a role in T cell activation-induced FasL expression. Inhibition of NF-kappaB activity in a T cell hybridoma leads to decreased FasL expression and apoptosis upon T cell receptor stimulation. We identified the NF-kappaB site in the FasL promoter that contributes to such regulation. Co-expression of p65 (Rel A) with the FasL promoter enhanced its activity, and co-expression of IkappaB dramatically inhibited the inducible promoter activity. In contrast, the transcription factor AP-1 is not required for activation-induced FasL promoter activity. These results define a role for NF-kappaB in mediating FasL expression during T cell activation. PMID: 9873041 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Biol Chem. 1999 Jan 1;274(1):376-83. Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation. Hall AJ, Vos HL, Bertina RM. Haemostasis and Thrombosis Research Centre, University Hospital Leiden, Leiden, 2300 RC The Netherlands. a.j.hall@sheffield.ac.uk Tissue Factor (TF) gene expression is transiently induced in human monocytic THP-1 cells by lipopolysaccharide (LPS). We characterized the transcription factor complexes binding to the TF gene promoter LPS response element (LRE) (-220 to -172), which contains binding sites for nuclear factor kappaB (NFkappaB) and activator protein 1 (AP1) transcription factors, and examined the nature of the activation of these factors during a 24-h time course of LPS stimulation. We found proteolysis of the cytoplasmic inhibitory protein IkappaBalpha and nuclear translocation of the NFkappaB/Rel family proteins p65 and c-Rel, corresponding to the transient binding of a p65/c-Rel heterodimer to the kappaB-like site of the LRE. AP1 binding to the LRE was found to be constitutive, with the majority of the AP1 complexes being JunD/Fra-2 heterodimers. A change in the activation state of the AP1 complexes was, however, found to be transient, as determined by JunD phosphorylation of AP1 bound to the proximal binding site. This directly correlates to the transient activation of Jun N-terminal kinase (SAPK/JNK). These data indicate that LPS induction of TF gene expression in monocytic THP-1 cells is regulated by both the transient phosphorylation of Jun-family proteins and the nuclear translocation and transient binding of NFkappaB/Rel proteins. PMID: 9867853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: 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] --------------------------------------------------------------- 55: Curr Opin Genet Dev. 1998 Oct;8(5):552-9. Combinatorial transcription factors. Wolberger C. Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA. cynthia@groucho.med.jhmi.edu Combinatorial regulation of eukaryotic transcription is mediated by proteins that associate in a specific manner to form multiprotein DNA-bound complexes. Substantial progress has recently been made towards the understanding of the molecular determinants of the protein-protein and protein-DNA interactions that govern assembly of these complexes. Three-dimensional structures have been determined of the MATalpha2/MCM1-DNA complex, the p50/p65 Rel homology domain heterodimer bound to DNA, the NFAT/Fos-Jun/DNA quaternary complex, and of the GABPalpha/beta ETS domain-ankyrin repeat heterodimer bound to DNA. Publication Types: Review Review, Tutorial PMID: 9794820 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: J Exp Med. 1998 Oct 5;188(7):1381-4. Stimulus-dependent synergism of the antiapoptotic tumor necrosis factor receptor-associated factor 2 (TRAF2) and nuclear factor kappaB pathways. Lee SY, Kaufman DR, Mora AL, Santana A, Boothby M, Choi Y. Department of Pathology, Hallym Medical School, Choonchun, Kangwon-do, 200-702, Korea. Tumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic signaling pathways. Thus, although TNF can cause apoptosis and may prove useful in the treatment of malignancies, most cells are resistant to TNF-induced cell death unless de novo protein synthesis is inhibited. Previous studies suggested that TNF activation of the nuclear factor (NF)-kappaB transcription factor family antagonizes the proapoptotic signals initiated by TNF-alpha. TNF receptor-associated factor (TRAF)2 has also been shown to mediate crucial antiapoptotic signals during TNF stimulation, yet is not essential in activation of NF-kappaB under physiologic conditions, thus raising questions about the relationship between these antiapoptotic pathways. We report here that inhibition of TRAF2 and NF-kappaB function in primary cells, by coexpression of a constitutive repressor of multiple NF-kappaB/Rel proteins (IkappaBalpha.DN) and a dominant negative form of TRAF2 (TRAF2.DN), synergistically enhanced TNF-induced apoptosis. The effects were stimulus dependent, such that neither inhibitory molecule affected Fas- and daunorubicin-induced apoptosis to the same degree as TNF-induced death. These findings indicate that the NF-kappaB and TRAF2 pathways activate independent antiapoptotic mechanisms which act in concert to suppress the proapoptotic signals induced by TNF-alpha. PMID: 9763618 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: J Immunol. 1998 Oct 1;161(7):3464-8. Role of nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways in IL-1 beta-mediated induction of alpha-PDGF receptor expression in rat pulmonary myofibroblasts. Lindroos PM, Rice AB, Wang YZ, Bonner JC. Airway Inflammation Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Induction of the alpha-platelet-derived growth factor receptor (PDGF-Ralpha) by IL-1beta in lung myofibroblasts enhances mitogenic and chemotactic responses to PDGF, and this could be a mechanism of myofibroblast hyperplasia during lung fibrogenesis. Since the regulation of many genes by IL-1beta involves activation of NF-kappaB and mitogen-activated protein (MAP) kinases, we examined these signaling pathways in the control of PDGF-Ralpha expression by IL-1beta in cultured rat lung myofibroblasts. Treatment of cells with pyrrolidine dithiocarbamate (PDTC), an antioxidant that inhibits NF-kappaB activation, completely blocked PDGF-Ralpha up-regulation by IL-1beta as assayed by [125I]PDGF-AA binding and PDGF-Ralpha mRNA expression, suggesting a role for NF-kappaB. However, while IL-1beta and TNF-alpha both induced nuclear binding of the Rel proteins p50 and p65 to an NF-kappaB consensus oligonucleotide in gel shift assays and caused transient degradation of inhibitor of NF-kappaB-alpha (IkappaB-alpha) in the cytoplasm of myofibroblasts, only IL-1beta upregulated PDGF-Ralpha. These results suggest that NF-kappaB activation alone is not sufficient for up-regulation of PDGF-Ralpha. An investigation of MAP kinase signaling pathways revealed that IL-1beta or PDTC activated extracellular signal-regulated kinase-2 (ERK-2) and c-jun NH2 terminal kinase-1 (JNK-1) phosphorylation of PHAS-1 and c-Jun substrates, respectively. Pretreatment of cells with the MAP kinase kinase-1 (MEK1) inhibitor PD 98059 blocked IL-1beta-induced activation of ERK-2 by more than 90% but enhanced IL-1beta-stimulated induction of PDGF-Ralpha expression fourfold. Taken together, these data suggest that IL-1beta activates both positive and negative signaling pathways that control the expression of PDGF-Ralpha. IL-1beta appears to mediate its negative effects on PDGF-Ralpha expression via MAP kinase activation, while the factor(s) that mediate induction of PDGF-Ralpha remain to be elucidated. PMID: 9759865 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Int J Cancer. 1998 Sep 25;78(1):16-20. Peripheral T lymphocytes from women with breast cancer exhibit abnormal protein expression of several signaling molecules. Kurt RA, Urba WJ, Smith JW, Schoof DD. Laboratory of Cellular Immunology, Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, OR, USA. Robert_Kurt@phsor.org We examined signaling molecules of peripheral blood T lymphocytes obtained from women with breast cancer. In 6 of 14 patients, T lymphocytes displayed an impaired ability to translocate NFeB p65 (Rel-A) following activation by anti-CD3 and IL-2. This observation was made despite normal cytoplasmic levels of the Rel-A protein. We also detected abnormally low levels of the signaling molecules T-cell receptor (TCR)-zeta, ZAP-70 and p56lck in 4 of 14 breast cancer patients, i.e., defects in T-cell signaling molecules. T lymphocytes from 6 of the 14 patients also exhibited an increased expression of the dual specificity phosphatase, map kinase phosphatase-1 (MKP-1). MKP-1 inactivates MAP kinase and therefore may interfere with the activation of c-jun and c-fos. Abnormalities of I or more signaling molecules were found in 9 of 14 patients; however, only 3 patients had T cells that exhibited all 5 defects. Our data have implications for the detection of potentially dysfunctional T cells in patients with cancer. For example, the analysis of only 1 signaling molecule may allow patients with significant defects in T-cell signaling to go unnoticed. Finally, despite impaired Rel-A translocation, T cells were capable of transcribing IL-2. Impairments in the translocation of Rel-B and c-Rel further suggest that the NFKB family members Rel-A, Rel-B and c-Rel are not required for the transcription of IL-2 in the peripheral T lymphocytes of patients with breast cancer. PMID: 9724088 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: J Biol Chem. 1998 Aug 28;273(35):22201-8. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites. Marks-Konczalik J, Chu SC, Moss J. Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590, USA. marksj@gwgate.nhlbi.nih.gov The involvement of AP-1 and NF-kappaB transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kappaB sites, in a hiNOS promoter extending from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment with a cytokine mixture (CM) containing interleukin 1-beta, interferon-gamma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad located -5301 base pairs upstream decreased gene activation by 90% in a -8.3-kb promoter and a shorter -5.574-kb promoter. Disruption of AP-1 (at -5115) or NF-kappaB (at -115 and -8283) sites reduced promoter activity by 45, 67, and 52%, respectively. Responsiveness to CM was decreased by 85% in constructs mutated in both NF-kappaB sites. By gel retardation analyses, CM increased AP-1- and NF-kappaB binding. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Each kappaB site bound different complements of NF-kappaB/Rel family members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). Rel A was maximally, whereas IkappaB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kappaB inding activity. Thus, AP-1 and NF-kappaB are important cis-elements for induction of hiNOS gene transcription. PMID: 9712833 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Mol Cell. 1998 Mar;1(4):543-51. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1. Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi A, Green DR. Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA. Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL. PMID: 9660938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: J Mol Med. 1998 Jun;76(7):480-9. Comment in: J Mol Med. 1998 Jun;76(7):459-60. Transcriptional cross-talk, the second mode of steroid hormone receptor action. Gottlicher M, Heck S, Herrlich P. Forschungszentrum Karlsruhe, Institute of Genetics, Germany. Physiological and therapeutic activities of glucocorticoids and other steroid hormones are mediated by the family of steroid hormone receptors. In addition to the classical mode of receptor action which involves binding as a dimer to regulatory sequences in target gene promoters and subsequent activation of transcription, a second mode of action is based predominantly on protein-protein interactions. As the paradigm of this so-called transcriptional cross-talk, the glucocorticoid receptor (GR) and the AP-1 transcription factor interact on target gene promoters which contain only a binding site for either one of the two transcription factors. Most frequently negative interference of both factors with each other's activity has been observed, for example, when AP-1 is composed of c-Fos and c-Jun; however, synergism is also possible under cell-specific conditions and when AP-1 is a homodimer of c-Jun. Since the detection of the GR/AP-1 cross-talk numerous other examples of transcription factor interactions have been described. Many members of the nuclear hormone receptor superfamily, including class II receptors, have been shown to participate in such cross-talk. Moreover, the transcription factor families of NF-kappaB/Rel as well as Stat, Oct, and C/EBP are engaged in cross-talk with steroid receptors. Despite the identification of a multitude of target genes which appear to be regulated by this type of transcription factor interaction, the exact molecular mechanism of the cross-talk has not yet been elucidated. This review discusses the current models to explain the molecular events of transcription factor cross-talk. Concepts are emphasized which suggest that the classical and the cross-talk mode of steroid receptor action can be triggered separately by the choice of specific ligands. A final section summarizes the partially contradictory data which assign a certain type of receptor action to a biological response particularly in the immune system. Publication Types: Review Review, Tutorial PMID: 9660166 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: 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] --------------------------------------------------------------- 63: Blood. 1998 Apr 15;91(8):2857-65. Retinoic acid selectively inhibits lipopolysaccharide induction of tissue factor gene expression in human monocytes. Oeth P, Yao J, Fan ST, Mackman N. Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA. Expression of tissue factor (TF) by activated monocytes in several diseases leads to disseminated intravascular coagulation. Lipopolysaccharide (LPS)-induced monocyte TF expression is downregulated by the nuclear hormone all-trans retinoic acid (ATRA). In this study, we examined the mechanism by which ATRA inhibits monocyte TF expression. We show that ATRA selectively inhibited LPS induction of TF expression in human monocytes and monocytic THP-1 cells without affecting LPS induction of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8). Inhibition of TF expression occurred at the level of transcription as determined by nuclear run-on. ATRA did not significantly alter the binding or functional activity of the transcription factors c-Fos/c-Jun and c-Rel/p65, which are required for LPS induction of the TF promoter in monocytic cells. In contrast to the ATRA inhibition of the endogenous TF gene, LPS induction of the cloned TF promoter was not inhibited by ATRA in transiently transfected THP-1 cells. Our results demonstrate that ATRA selectively inhibited LPS-induced TF gene transcription in human monocytic cells by a mechanism that does not involve repression of AP-1- or NF-kappaB-mediated transcription. PMID: 9531596 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: J Immunol. 1997 Dec 1;159(11):5463-73. Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1. Castellanos MC, Munoz C, Montoya MC, Lara-Pezzi E, Lopez-Cabrera M, de Landazuri MO. Servicio de Inmunologia, Universidad Autonoma de Madrid, Spain. The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1. PMID: 9580241 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: 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] --------------------------------------------------------------- 66: Mol Cell Biol. 1998 May;18(5):2997-3009. AP-1 factors play an important role in transformation induced by the v-rel oncogene. Kralova J, Liss AS, Bargmann W, Bose HR Jr. Department of Microbiology and the Institute for Cellular and Molecular Biology, University of Texas at Austin, 78712-1095, USA. v-rel is the oncogenic member of the Rel/NF-kappaB family of transcription factors. The mechanism by which v-Rel induces transformation of avian lymphoid cells and fibroblasts is not precisely known. However, most models propose that v-rel disrupts the normal transcriptional regulatory network. In this study we evaluated the role of AP-1 family members in v-Rel-mediated transformation. The overexpression of v-Rel, c-Rel, and c-Rel delta resulted in a prolonged elevation of c-fos and c-jun expression and in a sustained repression of fra-2 at both the mRNA and protein levels in fibroblasts and lymphoid cells. Moreover, the transforming abilities of these Rel proteins correlated with their ability to alter the expression of these AP-1 factors. v-Rel exhibited the most pronounced effect, whereas c-Rel, with poor transforming ability, elicited only moderate changes in AP-1 levels. Furthermore, c-Rel delta, which exhibits enhanced transforming potential relative to c-Rel, induced intermediate changes in AP-1 expression. To directly evaluate the role of AP-1 family members in the v-Rel transformation process, a supjun-1 transdominant mutant was used. The supjun-1 mutant functions as a general inhibitor of AP-1 activity by inhibiting AP-1-mediated transactivation and by reducing AP-1 DNA-binding activity. Coinfection or sequential infection of fibroblasts or lymphoid cells with viruses carrying rel oncogenes and supjun-1 resulted in a reduction of the transformation efficiency of the Rel proteins. The expression of supjun-1 inhibited the ability of v-Rel transformed lymphoid cells and fibroblasts to form colonies in soft agar by over 70%. Furthermore, the expression of supjun-1 strongly interfered with the ability of v-Rel to morphologically transform avian fibroblasts. This is the first report showing that v-Rel might execute its oncogenic potential through modulating the activity of early response genes. PMID: 9566919 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: Oncogene. 1997 Nov 27;15(22):2675-85. Identification and characterization of R-ras3: a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution. Kimmelman A, Tolkacheva T, Lorenzi MV, Osada M, Chan AM. The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA. Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins. PMID: 9400994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: 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] --------------------------------------------------------------- 69: 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] --------------------------------------------------------------- 70: Proc Natl Acad Sci U S A. 1997 May 13;94(10):4919-24. Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1. Sun LJ, Peterson BR, Verdine GL. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA. The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein-protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors. PMID: 9144165 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Leukemia. 1997 Apr;11 Suppl 3:402-4. v-Rel activates the proto-oncogene c-Jun promoter: a correlation with its transforming activity. Fujii M, Minamino T, Nomura M, Miyamoto K, Tanaka J, Seiki M. Department of Immunotherapeutics, Tokyo Medical and Dental University, Japan. v-Rel is a transforming protein of the reticuloendotheliosis virus, and is a transcription factor regulating various cellular genes. We found that v-Rel activates the promoter of the proto-oncogene c-jun in a transient transfection assay system. Moreover, the expression of endogenous c-jun was augmented in cells expressing exogenous v-Rel, but not c-Rel. The transcriptional activities of v-Rel to the tested promoters containing the kB-site are lower than that of c-Rel, but that to the c-jun promoter was much higher than that of c-Rel. The N-terminal DNA binding domain of v-Rel, which is responsible for its high transforming activity of v-Rel was also responsible for the high transcriptional activity to the c-jun promoter. Thus, the activity of v-Rel upon the c-jun promoter correlates well with its transforming ability. Since c-Jun plays pivotal roles on cell proliferation in various types of cells, the activation of c-jun expression by v-Rel may be an essential step for the oncogenic transformation caused by v-Rel. PMID: 9209405 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Mol Cell Biol. 1997 Mar;17(3):1314-23. Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding. Parra E, Varga M, Hedlund G, Kalland T, Dohlsten M. Department of Cell and Molecular Biology, University of Lund, Sweden. We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively. PMID: 9032258 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Arterioscler Thromb Vasc Biol. 1997 Feb;17(2):365-74. Regulation of the tissue factor gene in human monocytic cells. Role of AP-1, NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. Oeth P, Parry GC, Mackman N. Department of Immunology, Scripps Research Institute, La Jolla, Calif. 92037, USA. Tissue factor (TF) expression by peripheral blood monocytes during sepsis initiates intravascular thrombosis. Bacterial lipopolysaccharide (LPS) rapidly induces TF gene transcription in monocytes. The human TF promoter contains binding sites for the transcription factors AP-1, c-Rel/p65, Egr-1, and Sp1. NF-kappa B/Rel proteins have been shown to physically interact with both AP-1 and Sp1 proteins. In this study, we investigated the role of these transcription factors in uninduced and LPS-induced TF gene expression in human monocytic THP-1 cells. Deletional analysis indicated that five Sp1 sites mediated basal expression in uninduced cells. The two AP-1 sites bound c-Fos/c-Jun heterodimers in both unstimulated and LPS-stimulated cells. Maximal LPS induction of the TF promoter required the two AP-1 sites and the kappa B site within the LPS response element. Disruption of the conserved spacing between the proximal AP-1 site and the kappa B site abolished LPS induction. Replacement of the two AP-1 sites with intrinsically bent DNA partially restored LPS induction, suggesting an additional structural role for the AP-1 sites. Synergistic transactivation of the LPS response element in Drosophila Schneider cells by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65. These data indicated that c-Fos/c-Jun, c-Rel/p65, and Sp1 regulate TF gene expression in human monocytic cells. PMID: 9081693 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Annu Rev Immunol. 1997;15:707-47. Transcription factors of the NFAT family: regulation and function. Rao A, Luo C, Hogan PG. Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA. arao@cbr.med.harvard.edu As targets for the immunosuppressive drugs cyclosporin A and FK506, transcription factors of the NFAT (nuclear factor of activated T cells) family have been the focus of much attention. NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a primary target for inhibition by cyclosporin A and FK506. Calcineurin controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells by interacting with an N-terminal regulatory domain conserved in the NFAT family. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability to bind to certain regulatory elements in cytokine genes. NFAT is also notable for its ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun) family to composite NFAT:AP-1 sites, found in the regulatory regions of many genes that are inducibly transcribed by immune-system cells. This review discusses recent data on the diversity of the NFAT family of transcription factors, the regulation of NFAT proteins within cells, and the cooperation of NFAT proteins with other transcription factors to regulate the expression of inducible genes. Publication Types: Review PMID: 9143705 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: 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] --------------------------------------------------------------- 76: EMBO J. 1996 Jul 15;15(14):3744-50. Activation of the IL-2 gene promoter by HTLV-I tax involves induction of NF-AT complexes bound to the CD28-responsive element. Good L, Maggirwar SB, Sun SC. Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, PA 17033, USA. The tax gene product of the type I human T-cell leukemia virus (HTLV-I) is a potent transcriptional activator of various growth-related cellular genes, including that encoding interleukin-2 (IL-2). Tax activation of many of these target genes appears to be mediated by the NF-kappa B/Rel and CREB/ATF family of cellular transcription factors. However, the mechanism by which Tax transactivates the IL-2 gene remains unclear. In the present study, we demonstrate that neither NF-kappa B/Rel nor CREB/ATF is sufficient for Tax-mediated activation of the IL-2 promoter. Two novel nuclear protein complexes are induced by Tax and specifically bind to an IL-2 gene enhancer, the CD28-responsive element (CD28RE). Immunobiochemical analyses suggest that these DNA binding complexes contain at least two members of the nuclear factor of activated T cells, NF-ATp and NF-ATc. However, the CD28 binding NF-AT complexes do not contain Jun and Fos family proteins that have been proposed to serve as NF-AT partners in the activation of the IL-2 NF-AT motif. Transient transfection studies demonstrate that the in vivo expressed NF-ATp binds to the CD28RE probe and enhances Tax-mediated activation of this critical IL-2 enhancer. We demonstrate further that binding of NF-AT to CD28RE is critical for Tax activation of the IL-2 promoter. Together, these results suggest a novel mechanism of Tax-mediated activation of the IL-2 gene, which involves the induction of NF-AT-containing CD28RE binding complexes. PMID: 8670878 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: J Leukoc Biol. 1996 Jul;60(1):8-26. Endotoxin signal transduction in macrophages. Sweet MJ, Hume DA. Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia. Through its action on macrophages, bacterial lipopolysaccharide (LPS) or endotoxin can trigger responses that are protective or injurious to the host. This review examines the effects of LPS on macrophages by following events from the cell surface to the nucleus. The involvement of protein tyrosine kinases, mitogen-activated protein kinases, protein kinase C, G proteins, protein kinase A, ceramide-activated protein kinase, and microtubules in this process are reviewed. At the nuclear level, rel, C/EBP, Ets, Egr, fos, and jun family members have been implicated in activation of LPS-inducible gene expression. Publication Types: Review PMID: 8699127 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Oncogene. 1996 Jun 20;12(12):2595-604. Synergistic stimulation of avian I kappa B alpha transcription by rel and fos/jun factors. Kralova J, Schatzle JD, Liss AS, Bargmann W, Bose HR Jr. Department of Microbiology and the Cell Research Institute, University of Texas at Austin, Austin, TX 78712-1095, USA. Rel/NF-kappa B transcription factors and I Kappa B alpha function in an autoregulatory network. Avian I kappa B alpha transcription is increased in response to both c-Rel and v-Rel. This study shows that I kappa B alpha transcription is synergistically stimulated by Rel and AP-1 factors (c-Fos and c-Jun). However, the response to v-Rel and the AP-1 factors was not as vigorous as that of c-Rel and AP-1. A 386 bp region of the I kappa B alpha promoter (containing two NF-kappa B and one AP-1 binding sites) was shown to be both necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1 proteins. In addition, an imperfect NF-kappa B binding site was found to overlap the AP-1 binding site. Mutation of either of the NF-kappa B binding sites or the AP-1 binding site dramatically decreased the response of the I kappa B alpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of c-Rel or v-Rel resulted in the formation of DNA binding complexes associated with the imperfect NF-kappa B binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappa B site stronger than c-Rel, and overexpression of v-Rel also resulted in the formation of a v-Rel containing complex bound to a consensus AP-1 site. These studies address the difference in c-Rel and v-Rel's ability to synergistically stimulate I kappa B alpha expression in conjunction with the AP-1 factors. PMID: 8700518 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Oncogene. 1996 May 16;12(10):2193-202. Selective activation of the proto-oncogene c-jun promoter by the transforming protein v-Rel. Fujii M, Minamino T, Nomura M, Miyamoto KI, Tanaka J, Seiki M. Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Japan. The transcription factor v-Rel is a transforming protein of the reticuloendotheliosis virus. We found that v-Rel activates the promoter of the proto-oncogene c-jun. Two elements in the c-jun promoter were required for the activation by v-Rel. One was a kB-site (v-Rel binding site), and the other was a c-jun promoter region between -52 and +148 (c-jun promoter (-52/+148)). Two promoters with the kB-site(s), those of human immunodeficiency virus (HIV) and SV40, were not activated by v-Rel, but their kB-sites were activated when introduced upstream of the c-jun promoter (-52/+148). Thus, the c-jun promoter (-52/+148) had information for the selective activation of the c-jun promoter by v-Rel. v-Rel bound to the c-jun kB-site with the higher affinity than c-Rel, thereby activating the c-jun promoter more efficiently than c-Rel. Moreover, the activity of v-Rel mutants upon the c-jun promoter correlates with their transforming activity. Thus, the c-jun promoter activation by v-Rel may play a role in the transformation caused by v-Rel. PMID: 8668346 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: J Biol Chem. 1996 Apr 12;271(15):8971-6. Interaction between c-Rel and the mitogen-activated protein kinase kinase kinase 1 signaling cascade in mediating kappaB enhancer activation. Meyer CF, Wang X, Chang C, Templeton D, Tan TH. Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. The Rel family of transcription factors are important mediators of various cytokine stimuli such as interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and CD28 costimulation in T cell effector responses. These stimuli induce Rel family DNA-binding activity to the kappaB enhancer and CD28 response elements of many cytokine gene promoters leading to cytokine production. Consistent with the importance of Rel family induction during immune responses, c-Rel knockout mice exhibit profound defects in T cell functions including IL-2 secretion and T cell proliferative responses to CD28 plus T cell receptor costimulation. The novel protein kinases, c-Jun NH2-terminal kinases (JNKs)/stress-activated protein kinases, are also activated by TNF-alpha, IL-1, and CD28 costimulation. Because of the common regulation of c-Rel and JNK1 by these agents in T cells, we investigated the role of JNK1 in c-Rel activation. We found that MAP kinase kinase kinase (MEKK) 1, a JNK1 activator, induced transcription from the human immunodeficiency virus-1 long terminal repeat and IL-2R alpha promoters in a kappaB-dependent manner. Coexpression of IkappaBalpha, a c-Rel inhibitor, inhibited the MEKK1-induced transcriptional activity. JNK1 synergized with MEKK1 in activating transcription from a kappaB-driven heterologous promoter. Furthermore, JNK1 associated with c-Rel in vivo in Jurkat T cells by coimmunoprecipitation assays and bound directly to c-Rel in a yeast two-hybrid assay. c-Rel also competed with c-Jun in in vitro kinase assays. However, JNK1 did not phosphorylate c-Rel, NF-kappaB, and IkappaB alpha in vitro, indicating that c-Rel may serve as a docking molecule to allow JNK1 phosphorylation of certain Rel-associated proteins. Transactivation of the IL-2Ralpha and HIV-kappaB-driven promoters by c-Rel was augmented by coexpression of MEKK1. These results demonstrate the first significant role for the MEKK1 kinase cascade module in c-Rel-mediated transcription. PMID: 8621542 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Haemostasis. 1996;26 Suppl 1:17-9. Regulation of tissue factor gene expression in human monocytic and endothelial cells. Mackman N. Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. Tissue factor (TF) is inducibly expressed within the vasculature by monocytes and endothelial cells. Transcriptional activation of the TF gene in these two cell types by bacterial lipopolysaccharide (LPS) or cytokines appears to be regulated by a common mechanism. Functional studies identified a 56-bp LPS response element which contains two AP-1 sites and a kappaB site. Assembly of a multiprotein complex composed of Fos-Jun and c-Rel-p65 heterodimers is required to induce TF gene transcription. Inhibiting proteolytic degradation of IkappaBalpha prevents nuclear translocation of c-Rel-p65 heterodimers and blocks inducible TF expression in monocytic and endothelial cells. Publication Types: Review Review, Tutorial PMID: 8904167 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: Drug Metab Dispos. 1996 Jan;24(1):15-22. Cytochrome P4502E1- and cytochrome P4502B1/2B2-catalyzed carbon tetrachloride metabolism: effects on signal transduction as demonstrated by altered immediate-early (c-Fos and c-Jun) gene expression and nuclear AP-1 and NF-kappa B transcription factor levels. Gruebele A, Zawaski K, Kaplan D, Novak RF. Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48201-2654, USA. We have previously reported that carbon tetrachloride (CCI4) stimulates c-fos, c-jun, and Ca(2+)-activated neutral protease gene expression in rat hepatic tissue (Zawaski et al., Biochem. Biophys. Res. Comm. 197, 585-590, 1993). The proteins c-Fos and c-Jun constitute inducible transcription factors in signal transduction and regulate the transcriptional activation of a battery of genes involved in cell growth and division. The present study was initiated to characterize the role of cytochrome P450 expression and metabolic activation on the magnitude of immediate-early (i.e. c-fos and c-jun) gene expression. Animals were treated either with diallyl sulfide, N-acetylcysteine, pyridine, or phenobarbital before treatment with CCI4. Total and poly(A)+ RNA were isolated, and c-fos and c-jun mRNA levels were analyzed by Northern blot and reverse transcriptase-polymerase chain reaction analyses. Treatment of animals with CCI4 increased c-fos and c-jun mRNA levels from below the limit of detection in control tissue to intense bands within 30 min of treatment, with maximal expression monitored at 1 and 2 hr posttreatment. Treatment of animals with diallyl sulfide alone also elevated c-fos and c-jun mRNA expression to detectable levels. However, pretreatment of animals with diallyl sulfide before treatment with CCI4 produced a 76-92% decrease in c-fos and c-jun mRNA levels, relative to that monitored for CCI4-treated animals. Pretreatment with N-acetylcysteine did not affect c-fos or c-jun mRNA levels and diminished CCI4-stimulated c-fos and c-jun gene expression by 44 and 55%, respectively, relative to the immediate-early gene mRNA levels monitored in the hepatic tissue of CCI4-treated animals. Pretreatment of animals with the CYP2E1 inducer pyridine for 24 hr had only a marginal effect on c-fos mRNA levels, but increased CCI4-stimulated c-fos and c-jun mRNA levels by an additional approximately 2- to approximately 4-fold over those monitored in the uninduced hepatic tissue of CCI4-treated animals. Whereas phenobarbital treatment alone enhanced c-fos expression only marginally, CCI4 treatment of phenobarbital-pretreated animals increased c-fos expression by up to an additional approximately 8-fold and c-jun mRNA levels by up to an additional approximately 5-fold over the respective levels monitored in the hepatic tissue of CCI4-treated animals. Enhanced CYP2E1 or CYP2B1/2B2 levels after treatment with pyridine or phenobarbital elevated c-fos mRNA over untreated controls. This increase was marginal, however, and detectable only with reverse transcriptase-polymerase chain reaction. Examination of nuclear levels of the heterodimeric c-Fos and c-Jun AP-1 transcription factor complex revealed a time-dependent increase in AP-1 levels. AP-1 transcription factor binding was confirmed using competitor consensus sequences and antibody supershifts. Nuclear levels of NF-kappa B, a transcription factor complex implicated in hepatocyte proliferation and apoptotic or programmed cell death, were also examined. NF-kappa B, which consists of the p50 and p65/Rel A polypeptides, was increased in hepatic nuclear extracts at 2 and 24 hr after CCI4 administration, with a concomitant decrease in the p50 polypeptide. Thus, the magnitude of CCI4 stimulation of the immediate-early genes c-fos and c-jun is dependent on metabolic activation by the P450s, and the magnitude of the effect is dependent on the levels and isozyme composition of P450s in the tissue. Furthermore, nuclear transcription factor levels of AP-1 and NF-kappa B are elevated in response to this toxicant. PMID: 8825185 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Biochem Biophys Res Commun. 1995 Dec 14;217(2):501-8. The transformation of c-jun-overexpressing cells is correlated with IGFS-induced c-jun phosphorylation. Lagarrigue S, Heberden C, Martel P, Gaillard-Sanchez I. Laboratoire de Nutrition et Securite Alimentaire-INRA, Jouy en Josas, France. We have analyzed, in parental and c-jun-transfected rat liver epithelial (REL) cells, the capacity of the insulin-like growth factors (IGF1 and 2) to stimulate growth under anchorage dependent and independent conditions and the role of the c-jun protein in the response to these factors. Although the increase of the proliferation of cells in monolayer was similar for the two lines, only the c-jun-transfected cells formed colonies in soft agar after IGFs exposure. We showed that IGF1 and 2 did not modify the expression of c-fos or c-jun proteins during short-time exposures but both factors enhanced c-jun phosphorylation which suggests that this phenomenon could be involved in IGFs-regulated gene expression. Moreover, we showed that both the overexpression and the increase of phosphorylation status of c-jun protein were necessary to transform REL cells. PMID: 7503728 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Eur J Cancer B Oral Oncol. 1995 Nov;31B(6):384-91. Differential c-myc, c-jun, c-raf and p53 expression in squamous cell carcinoma of the head and neck: implication in drug and radioresistance. Riva C, Lavieille JP, Reyt E, Brambilla E, Lunardi J, Brambilla C. Lung and Airways Cancer Research Group, Albert Bonniot Institute, La Tronche, France. The expression of oncogenes c-myc, c-jun and c-raf and tumour suppressor gene p53 was assessed by northern blot analysis of 42 tumours and p53 protein expression by immunohistochemistry on paraffin-embedded sections from 36 specimens of squamous cell carcinoma of the head and neck (SCCHN) obtained before therapy. Of the 42 tumours, 89, 100 and 100% expressed c-myc, c-jun and c-raf oncogenes, respectively. These oncogene expressions did not correlate with sex, age or clinical stage of the disease. However, an association was found between low c-myc expression (P = 0.0001) and high c-jun expression (P = 0.0001) and absence of tumoral response to neoadjuvant chemotherapy. On the other hand, c-raf overexpression was observed in patients resistant to radiation therapy (P = 0.0494). Forty-two per cent of the tumours showed p53 protein overexpression, which did not correlate with any clinical parameter. This p53 protein overexpression was associated with high p53 mRNA levels (REL) (P = 0.0223). A correlation was found between increased c-myc RNA expression and lack of p53 protein expression (P = 0.0407). In addition, a lack of p53 protein expression was indicative of tumour relapse (P = 0.05). None of these biological parameters were associated with disease-free survival (Cox-Mantel test). In conclusion, the overexpression of c-myc, c-jun and c-raf may be independently associated to tumoral response to chemotherapy or radiotherapy, or to tumour relapse, but fail to predict long-term survival. PMID: 8746269 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: J Immunol. 1995 Aug 1;155(3):1132-40. Costimulation of human CD4+ T cells with LFA-3 and B7 induce distinct effects on AP-1 and NF-kappa B transcription factors. Parra E, Varga M, Sigvardsson M, Hedlund G, Kalland T, Leanderson T, Sjogren HO, Dohlsten M. Wallenberg Laboratory, Department of Tumor Immunology, University of Lund, Sweden. We have earlier shown that stimulation of human CD4+ T cells with SEA presented on Chinese hamster ovary (CHO)-DR transfectants coexpressing either B7 or LFA-3 resulted in distinct cytokine profiles. We now demonstrate that B7, but not LFA-3, strongly costimulated IL-2 transcription and mRNA expression in CD4+ T cells. Maximal increase in IL-2 transcription was recorded with CHO-DR/B7/LFA-3, suggesting a cooperative effect of B7 and LFA-3 at the transcriptional level. Gel-shift analysis demonstrated that stimulation of CD4+ T cells with CHO-DR and staphylococcal enterotoxin A was sufficient to induce significant amounts of NF-kappa B binding proteins, whereas induction of AP-1 binding proteins required costimulation. LFA-3 induced moderate levels of AP-1, but did not influence the levels of NF-kappa B, while B7 costimulation strongly induced both AP-1 and substantially enhanced NF-kappa B binding proteins. The CHO-DR/B7/LFA-3 triple transfectant induced a further increase in AP-1 and NF-kappa B binding proteins compared with the double transfectants. The level of Oct-1 binding proteins remained similar in all samples. Super-shift analysis revealed that the NF-kappa B complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The AP-1 binding proteins contained c-Jun, Jun-D, and Fra-1, but marginal amounts of Jun-B and c-Fos. Our results indicate distinct effects of B7 and LFA-3 costimulation on the activity of AP-1 and NF-kappa B. These may partly account for the differential effects of B7 and LFA-3 costimulation on IL-2 expression. PMID: 7543515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Immunobiology. 1995 Jul;193(2-4):128-36. Activation of NF-kappa B by the Tax protein of HTLV-1. Munoz E, Israel A. Unite de Biologie Moleculaire de l'Expression Genique, Institut Pasteur, Paris, France. The Tax protein, encoded by the human T cell leukemia virus HTLV-1, is responsible for transcriptional activation of the viral genome through conserved 21bp repeats located in its promoter. Tax also activates the transcription of cellular genes such as interleukin 2, interleukin 2 receptor (IL2R), GM-CSF, vimentin, c-fos, c-jun as well as the major histocompatibility complex class I genes. Tax does not bind DNA directly, but seems to activate transcription indirectly by enhancing the activity of the transcription factors that recognize responsive elements located in the promoters of the Tax-responsive genes, or by forming ternary complexes with these factors and DNA. One class of target sites for Tax are the kappa B sequences which are bound by members of the rel/NF-kappa B family. It has been previously shown that Tax is able to induce nuclear translocation of NF-kappa B. The activity of the NF-kappa B transcription factor is normally controlled through cytoplasmic retention by either of two types of molecules: the inhibitor I kappa B alpha/MAD3 or the p105 and p100 precursors of the p50 and p52 DNA-binding subunits. Treatment of cells with classical NF-kappa B inducers like TNF, IL-1, PMA or LPS results in MAD-3 degradation followed by nuclear translocation of NF-kappa B. On the other hand, the mechanisms involved in the dissociation of the cytoplasmic p105/p100-containing complexes are largely unknown. We demonstrate here that Tax can induce translocation of members of the NF-kappa B family retained in the cytoplasm through interaction with either p105 or p100. On the other hand Tax induces no apparent degradation of MAD-3. These results suggest that Tax activates NF-kappa B essentially through the p105/p100-retention pathway. Publication Types: Review Review, Tutorial PMID: 8530135 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: FASEB J. 1995 Jul;9(10):883-9. Regulation of the tissue factor gene. Mackman N. Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA. The tissue factor (TF) gene is expressed in a cell type-specific manner in vivo. It is constitutively expressed by several extravascular cell types and inducibly expressed within the vasculature by monocytes and endothelial cells. TF expression initiates thrombotic episodes associated with various diseases, including atherosclerosis, septic shock, and cancer. Regulatory elements within the human TF promoter have been identified by functional analysis of TF promoter-luciferase gene plasmids transiently transfected into various cell types. Transcription factors that control expression of the TF gene were identified using gel shift mobility assays. Induction of the TF gene in human monocytic cells and endothelial cells exposed to bacterial lipopolysaccharide or cytokines is mediated by a distal enhancer (-227 to -172 bp) containing two AP-1 sites and a kappa B site. Functional interactions between Fos-Jun heterodimers and c-Rel-p65 heterodimers are required for transcriptional activation of the TF gene. In contrast, serum and phorbol ester induction of the TF gene in human epithelial cells is controlled by a proximal enhancer (-111 to +14 bp) containing three overlapping Egr-1/Sp1 binding sites. Sp1 is constitutively expressed whereas Egr-1 expression is induced by serum or phorbol ester stimulation. Sp1 also mediates basal promoter activity. Thus, TF gene expression is complex and is regulated by a number of transcription factors that bind to distinct regions of the TF promoter. Publication Types: Review Review, Tutorial PMID: 7615158 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Arterioscler Thromb Vasc Biol. 1995 May;15(5):612-21. Transcriptional regulation of tissue factor expression in human endothelial cells. Parry GC, Mackman N. Department of Immunology, Scripps Research Institute, La Jolla, Calif. 92037, USA. Tissue factor (TF) expression by endothelial cells is implicated in thrombotic episodes in patients with a variety of clinical disorders. In a baboon model of lethal sepsis, TF is expressed by endothelial cells in the splenic microvasculature. In vitro, endothelial cells are induced to express TF in response to tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and bacterial endotoxin (lipopolysaccharide [LPS]). Here, we identified cis-acting regulatory elements that control TF gene transcription in primary human endothelial cells. Functional studies showed that the TF promoter contained a 56-bp enhancer (-227 to -172 bp), which included two activator protein-1 (AP-1) sites and a kappa B-like site, that mediated induction by TNF-alpha, IL-1 beta, and LPS. Electrophoretic mobility shift assays demonstrated that endothelial cells contained constitutive AP-1 binding activity, whereas the kappa B-like site, 5'-CGGAGTTTCC-3', bound an inducible nuclear complex composed of c-Rel-p65 heterodimers. Taken together, our data suggest that induction of TF gene transcription in endothelial cells is mediated by functional interactions between Fos-Jun and c-Rel-p65 heterodimers. PMID: 7749875 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Mol Cell Biol. 1995 May;15(5):2697-706. NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus. Masuda ES, Naito Y, Tokumitsu H, Campbell D, Saito F, Hannum C, Arai K, Arai N. Department of Cell Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304, USA. The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of cytokine genes. Here, we report the cDNA cloning, chromosomal localization, and initial characterization of a transcription factor related to NFATp and NFATc. The novel molecule, designated NFATx, exhibits in its middle a region very similar to the Rel homology domain in NFATc and NFATp. The amino-terminal region of NFATx also shows significant similarities to corresponding sequences in NFATc and NFATp and contains three copies of a conspicuous 17-residue motif of unknown function. We provide evidence showing that NFATx can reconstitute binding to the NFAT-binding site from the interleukin 2 promoter when combined with AP1 (c-Fos/c-Jun) polypeptides and that NFATx is capable of activating transcription of the interleukin 2 promoter in COS-7 cells when stimulated with phorbol ester and calcium ionophore. NFATx mRNA is preferentially and remarkably found in the thymus and at lower levels in peripheral blood leukocytes. The expression pattern of NFATx, together with its functional activity, strongly suggests that NFATx plays a role in the regulation of gene expression in T cells and immature thymocytes. PMID: 7739550 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: J Leukoc Biol. 1995 Apr;57(4):536-42. NFATp, a cyclosporin-sensitive transcription factor implicated in cytokine gene induction. Rao A. Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. The transcription factor NFATp (nuclear factor of activated T cells, preexisting) is likely to regulate the cyclosporin-sensitive transcription of cytokine genes and other activation-associated genes during the immune response. NFATp is the first identified member of a new family of transcription factors, whose DNA binding domains show a weak sequence similarity to those of the Rel (NF-kappa B) family of proteins. NFATp is expressed in several types of immune cells as a cytosolic protein that translocates to the nucleus following activation. The nuclear translocation is regulated by calcium and calcineurin and inhibited by cyclosporin A and FK506. In the nucleus, NFATp cooperates with Fos-Jun dimers and possibly with other transcription factors at composite elements in the regulatory regions of cytokine genes. The potential roles of NFATp and a related family member, NFATc, in cytokine gene transcription are discussed. Publication Types: Review Review, Tutorial PMID: 7722411 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: J Biol Chem. 1995 Feb 24;270(8):4138-45. A similar DNA-binding motif in NFAT family proteins and the Rel homology region. Jain J, Burgeon E, Badalian TM, Hogan PG, Rao A. Dana-Farber Cancer Institute, Department of Pathology, Boston, Massachusetts 02115. The cyclosporin-sensitive factor NFATp cooperates with Fos and Jun family proteins to regulate transcription of the interleukin 2 gene in activated T cells. We have defined a 187-amino-acid fragment of NFATp, located centrally within the protein sequence, as the minimal region required for DNA binding and for complex formation with Fos and Jun. The sequence of this region of NFATp shows a low degree of similarity to the Rel homology region. One specific short sequence in NFATp (RAHYETEG), located near the NH2 terminus of the DNA-binding domain, resembles a highly conserved sequence (RFRYxCEG) that is located near the NH2 terminus of the Rel homology region and that has been implicated in DNA binding by Rel family proteins. Mutational analysis demonstrates that the residues in this sequence that are identical in NFATp and Rel family proteins contribute to DNA binding by NFATp. Further, mutation of the threonine residue in this sequence to cysteine, as in Rel proteins, confers on NFATp a sensitivity to sulfhydryl modification similar to that of Rel family proteins. The results suggest that NFATp and Rel family proteins bind to DNA using similar structural motifs. PMID: 7876165 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: 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] --------------------------------------------------------------- 93: Cell Mol Biol Res. 1995;41(6):551-60. Suppression of oncogene-induced transformation by quercetin and retinoic acid in rat liver epithelial cells. Lagarrigue S, Chaumontet C, Heberden C, Martel P, Gaillard-Sanchez I. Laboratoire de Nutrition et Securite Alimentaire, INRA, Jouy-en-Josas, France. AP1 is a heterodimeric complex containing products of the Jun and Fos oncogene families. The c-fos and c-jun protooncogenes act as transcriptional activator for numerous cellular genes, and the overexpression of these genes may cause malignant transformation. In this study, to show evidence of a possible inhibition of AP1 transcriptional activity in molecular mechanisms of foodborne molecules, known to be negative modulators of carcinogenesis, we established two rat liver epithelial (REL) cell lines overexpressing either c-fos (43C line) or c-jun (RELcJ1 line) oncoproteins. Contrary to the 43C line, which was spontaneously transformed, the c-jun-transfected REL cells were only transformed in vitro after 12-O-tetra-decanoylphorbol 13-acetate (TPA) exposure. All trans-retinoic acid (RA) abolished the transformation of the 43C line and TPA-treated RELcJ1 cells, suggesting that RA could decrease AP1 activity in these cells despite c-fos or c-jun overexpression. Furthermore, we show for the first time that a flavonoid, quercetin, which is a natural component of vegetables, inhibited only the transformation of the 43C line. The spontaneous transformation of the c-fos-transfected REL cells was associated with the appearance of c-fos/AP1 complexes binding TRE, suggesting that c-fos/AP1 complexes are involved in the antitransforming mechanism of quercetin. PMID: 8777434 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Cell. 1994 Jun 17;77(6):795-8. NF-AT-AP-1 and Rel-bZIP: hybrid vigor and binding under the influence. Nolan GP. Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305. Publication Types: Review Review, Tutorial PMID: 8004669 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Biochem Biophys Res Commun. 1994 Jun 15;201(2):950-6. Stimulation of transcription factors NF kappa B and AP1 in endothelial cells subjected to shear stress. Lan Q, Mercurius KO, Davies PF. Department of Pathology, Pritzker School of Medicine, University of Chicago, IL 60637. Hemodynamic shear stress forces influence several important endothelial genes associated with arterial relaxation, pro/anti-coagulation, and growth control; however, the regulatory pathways remain unclear. Here we demonstrate stimulation of DNA binding activities of nuclear factor kappa B (NF kappa B), a member of the Rel Family of transcription factors, and nuclear factor activator protein-1 (AP-1) following exposure of endothelial cells to unidirectional shear stress in laminar flow. NF kappa B binding was stimulated within 30 minutes, reaching and maintaining maximal levels at 1 hour. DNA binding activity was inhibited by pre-incubation of nuclear extract with antibody directed against NF kappa B p65 subunit. AP-1 binding activity was biphasic, rising fourfold within 20 minutes and returning to basal levels before steadily increasing by 2 hours to a high level relative to basal values. These protein kinase C-coupled transcriptional factors may modulate endothelial genes that are shear stress-responsive and that possess appropriate binding sites in the promoter region. PMID: 8003036 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: J Biol Chem. 1994 Mar 18;269(11):8582-9. The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization. Okamoto S, Mukaida N, Yasumoto K, Rice N, Ishikawa Y, Horiguchi H, Murakami S, Matsushima K. Department of Pharmacology, Kanazawa University, Japan. FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13-acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the IL-8 promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (c-Rel, p65, p50, and p49). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which NF-kappa B binding was also decreased by FK506, indicating that both IL-8 kappa B-like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization. PMID: 7510691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: J Virol. 1994 Jan;68(1):276-88. Inducible and cell type-specific expression of VL30 U3 subgroups correlate with their enhancer design. Nilsson M, Bohm S. Center for Nutrition and Toxicology, Karolinska Institute, NOVUM, Huddinge, Sweden. The murine VL30 elements constitute one family of retrotransposons represented in 100 to 200 copies that are dispersed among the mouse chromosomes. On the basis of sequence homology, we have subdivided mouse VL30 members into four distinct U3 subgroups. The use of subgroup-specific probes in Northern (RNA) blot analyses shows that individual VL30 U3 subgroups are expressed in a tissue-specific manner. We show by in situ hybridization of mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) that VL30 expression is induced in epidermal keratinocytes but not in dermal fibroblasts. Transient transfections of reporter gene plasmids together with in vitro binding analysis indicate that TPA-induced VL30 transcription specific for keratinocytes is mediated by two cooperating sequence motifs in juxtaposed position. One sequence motif is shown to constitutively bind CREB- and Jun-related proteins in both keratinocytes and fibroblasts, whereas the other is a target for TPA-induced c-Rel/p65(NF-kappa B)-binding activity specifically in keratinocytes. These binding sites are found to be conserved within U3 subgroups and individual U3 regions showing induced expression in TPA-treated mouse epidermis. These results together with a sequence comparison between different U3 subgroups indicate that cell type-specific activity of transcription factors known to regulate VL30 transcription and the presence or absence of their cognate binding sites within individual U3 regions determine inducible and cell type-specific VL30 expression. The variable VL30 U3 regions might thus be useful tools to study inducible and cell type-specific transcription in many different cell systems. PMID: 8254739 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: EMBO J. 1993 Oct;12(10):3879-91. Cross-coupling of the NF-kappa B p65 and Fos/Jun transcription factors produces potentiated biological function. Stein B, Baldwin AS Jr, Ballard DW, Greene WC, Angel P, Herrlich P. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599. NF-kappa B and AP-1 represent distinct mammalian transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is typically composed of two DNA binding subunits, NF-kappa B p50 and NF-kappa B p65, which share structural homology with the c-rel proto-oncogene product. Similarly, the AP-1 transcription factor complex is comprised of dimers of the c-fos and c-jun proto-oncogene products or of closely related proteins. We now demonstrate that the bZIP regions of c-Fos and c-Jun are capable of physically interacting with NF-kappa B p65 through the Rel homology domain. This complex of NF-kappa B p65 and Jun or Fos exhibits enhanced DNA binding and biological function via both the kappa B and AP-1 response elements including synergistic activation of the 5' long terminal repeat of the human immunodeficiency virus type 1. These findings support a combinatorial mechanism of gene regulation involving the unexpected cross-coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity. PMID: 8404856 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: Mol Cell Biol. 1993 Aug;13(8):4609-17. Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro. Galang CK, Hauser CA. La Jolla Cancer Research Foundation, California 92037. The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity. PMID: 8101633 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: Eur J Biochem. 1993 Jan 15;211(1-2):7-18. Erratum in: Eur J Biochem 1993 Aug 1;215(3):907. The Ets family of transcription factors. Wasylyk B, Hahn SL, Giovane A. CNRS-LGME/INSERM-U. 184, Institut de Chimie Biologique, Faculte de Medecine, Strasbourg, France. Interest in the Ets proteins has grown enormously over the last decade. The v-ets oncogene was originally discovered as part of a fusion protein expressed by a transforming retrovirus (avian E26), and later shown to be transduced from a cellular gene. About 30 related proteins have now been found in species ranging from flies to humans, that resemble the vEts protein in the so-called 'ets domain'. The ets domain has been shown to be a DNA-binding domain, that specifically interacts with sequences containing the common core trinucleotide GGA. Furthermore, it is involved in protein-protein interactions with co-factors that help determine its biological activity. Many of the Ets-related proteins have been shown to be transcription activators, like other nuclear oncoproteins and anti-oncoproteins (Jun, Fos, Myb, Myc, Rel, p53, etc.). However, Ets-like proteins may have other functions, such as in DNA replication and a general role in transcription activation. Ets proteins have been implicated in regulation of gene expression during a variety of biological processes, including growth control, transformation, T-cell activation, and developmental programs in many organisms. Signals regulating cell growth are transmitted from outside the cell to the nucleus by growth factors and their receptors. G-proteins, kinases and transcription factors. We will discuss how several Ets-related proteins fit into this scheme, and how their activity is regulated both post- and pre-translationally. Loss of normal control is often associated with conversion to an oncoprotein. vEts has been shown to have different properties from its progenitor, which might explain how it has become oncogenic. Oncogene-related products have been implicated in the control of various developmental processes. Evidence is accumulating for a role for Ets family members in Drosophila development, Xenopus oocyte maturation, lymphocyte differentiation, and viral infectious cycles. An ultimate hope in studying transformation by oncoproteins is to understand how cells become cancerous in humans, which would lead to more effective treatments. vEts induces erythroblastosis in chicken. Cellular Ets-family proteins can be activated by proviral insertion in mice and, most interestingly, by chromosome translocation in humans. We are at the beginning of understanding the multiple facets of regulation of Ets activity. Future work on the Ets family promises to provide important insights into both normal control of growth and differentiation, and deregulation in illness. Publication Types: Review PMID: 8425553 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: Receptor. 1993 Spring;3(1):17-30. Identification of a specific pattern of "immediate-early" gene activation induced by estrogen during mitogenic stimulation of rat uterine cells. Cicatiello L, Sica V, Bresciani F, Weisz A. Istituto di Patologia generale e Oncologia, Prima Facolta di Medicina e Chirurgia, Universita di Napoli, Naples, Italy. Estrogen hormones are potent mitogens for certain target tissues, where they stimulate cell growth by inducing recruitment of quiescent cells in cycle and by fostering cell cycle progression. To define the molecular bases of the mitogenic action of these steroid hormones, the pattern of "immediate-early" gene expression was monitored during the early phases of estrogen stimulation of rat uterine cells in vivo. Nuclear run-on transcription and/or Northern blot RNA analysis indicate that c-jun, junB, jun-D, c-fos, TIS 1 (also called NGFI-B or nur/77) and TIS 8 (zif-268, krox24, egr-1, or NGFI-A) genes are all transiently activated in the uterus (up to 20-fold) within 30-120 min after treatment of adult ovariectomized rats with a mitogenic dose of 17b-estradiol. Conversely, JE gene mRNA accumulates progressively in estrogen-stimulated uterine cells, whereas TIS 11 and 21 genes are only slightly responsive to the hormone (less than twofold induction) and fos B,fra-1,fra-2,krox20 (egr-2), TIS 7 and 10, KC, and c-rel mRNAs are undetectable in rat uterus either before or after estrogen treatment. Stimulation in the presence of cycloheximide shows that only c-jun, jun-D, c-fos, and JE gene activations are primary responses to the hormone in rat uterine cells. These findings establish the direct mitogenic role of estrogen and identify for the first time a specific genetic program activated by these steroid hormones during stimulation of target cell proliferation. Furthermore, since most of the activated genes encode for transcription factors, these results enable us to envision how the mitogenic signal transmitted by the hormone can be elaborated and amplified within target cells by the products of estrogen-responsive genes, leading to a cascade of growth-dependent gene regulation, cell cycle progression, and, ultimately, cell division. PMID: 8348080 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: Tohoku J Exp Med. 1992 Oct;168(2):183-7. Possible involvement of nuclear oncoproteins in regulation of DNA replication. Asano M, Ishikawa H, Ito Y. Department of Viral Oncology, Institute for Virus Research, Kyoto University, Japan. Polyomavirus DNA replication requires its enhancer which contains an AP-1 site. We have shown that protooncogenes, c-jun and c-fos, whose products form heterodimeric transcription activator, AP-1, strongly stimulate polyomavirus DNA replication through the AP-1 site. The mechanisms by which this enhancer stimulates replication and transcription are different. By replacing the enhancer with the oligonucleotides representing the binding site of a transcription factor of interest, any transcription factor with the known binding sequence can be characterized in this replication assay. When Rel protein was examined, we were able to reveal a new domain in v-Rel protein which can stimulate replication strongly. Whether this domain also coincides with transforming potential of v-Rel protein is currently under investigation. Publication Types: Review Review, Tutorial PMID: 1339100 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: Curr Opin Cell Biol. 1992 Jun;4(3):496-501. Signal transduction: the nuclear target. Kerr LD, Inoue J, Verma IM. Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92186-5800. Fos and jun heterodimers activate the transcription of genes containing an AP-1 site. The activity of Fos and Jun proteins is regulated by post-translational modification. The activity of the rel/NF-kappa family of transcriptional factors is regulated by their sequestration in the cytoplasm in association with the inhibitor protein, I kappa B. An ankyrin repeat motif in I kappa B proteins is required for their direct association with rel/NF-kappa B. Publication Types: Review Review, Tutorial PMID: 1497922 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: Curr Opin Genet Dev. 1992 Feb;2(1):19-27. Crossed signals: oncogenic transcription factors. Forrest D, Curran T. Roche Institute of Molecular Biology, Department of Molecular Oncology and Virology, Nutley, New Jersey 07110. Transcription factors operate at key regulatory junctions in the cell's responses to diverse extracellular stimuli. We discuss how the transforming activity of mutated transcription factors encoded by several oncogenes (v-erbA, v-fos, v-jun and v-rel) may result in part from a loss of the integrated response to the signalling network. Publication Types: Review Review, Tutorial PMID: 1633422 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Nature. 1991 Aug 15;352(6336):635-8. Transformation suppressor activity of a Jun transcription factor lacking its activation domain. Lloyd A, Yancheva N, Wasylyk B. LGME-CNRS, U184-INSERM, Institut de Chimie Biologique, Faculte de Medecine, Strasbourg, France. The oncoprotein c-Jun is thought to be a mediator of ras transformation as both its synthesis and activity as a transcription factor are stimulated by ras expression. But c-Jun co-operates with ras in transformation assays, suggesting that they act along different pathways (reviewed in ref. 4). Here we show by means of a dominant-negative mutated transcription factor that c-Jun potentially in conjunction with other factors that interact with it is necessary for transformation by ras. The mutant Jun lacks an activation domain and blocks stimulation of transcription by several oncoproteins, including Ras, v-Src, polyoma middle T, c-Jun and c-Fos, as well as by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The inhibition is specific for motifs that bind Jun: activation of an NF-kappa B/Rel motif is not affected. This Jun mutant acts as an anti-oncogene in ras-transformed cells, generating non-transformed revertants that have acquired anchorage and density-dependent growth, as well as reduced tumorigenicity in vivo. Mutants of other transcription factors designed to inhibit transformation will enable us to study their role in signal transduction. PMID: 1907719 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: Cancer Cells. 1989 Oct;1(2):65-6. Nuclear oncogenes. Lamph WW. Salk Institute, San Diego, California 92138. PMID: 2518150 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------