1: Immunol Cell Biol. 2005 Nov 7; [Epub ahead of print] Perturbations in nuclear factor-kappaB or c-Jun N-terminal kinase pathways in pancreatic beta cells confer susceptibility to cytokine-induced cell death. Thomas HE, Angstetra E, Fernandes RV, Mariana L, Irawaty W, Jamieson EL, Dudek NL, Kay TW. St Vincent's Institute of Medical Research, Melbourne, Victoria, Australia. Summary Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines. PMID: 16277639 [PubMed - as supplied by publisher] --------------------------------------------------------------- 2: J Neurochem. 2005 Aug;94(3):850-61. Epub 2005 Jul 7. Human immunodeficiency virus type 1 envelope glycoprotein 120 induces cyclooxygenase-2 expression in neuroblastoma cells through a nuclear factor-kappaB and activating protein-1 mediated mechanism. Alvarez S, Serramia MJ, Fresno M, Munoz-Fernandez M. Laboratory Inmuno-Biologia Molecular, Hospital General Universitario Gregorio Maranon, Madrid, Spain. Induction of cyclooxygenase-2 (COX-2) in the brain of people infected with human immunodeficiency virus type 1 (HIV-1) has been proposed as a cause of cognitive impairment in AIDS dementia. Here, we have analyzed the molecular mechanism by which its induction takes place in neuroblastoma cells. The HIV-1 envelope protein gp120 was able to induce COX-2 mRNA and protein in several human neuroblastoma cell lines, which express CXCR4 and CCR5 but not CD4. Moreover, gp120 induces COX-2 promoter transcription. Sequential deletions of the promoter show that deletion of a distal nuclear factor-kappaB (NF-kappaB) site abrogated gp120-dependent transcription. More importantly, overexpression of NF-kappaB inhibitory subunit, IkappaBalpha, completely abrogated gp120-induced COX-2 activity. However, transfection of p65/relA NF-kappaB was not enough to induce COX-2 transcription, suggesting that NF-kappaB was necessary but not sufficient to control COX-2 transcription induced by gp120. In addition to NF-kappaB, activating protein-1 (AP-1) but not nuclear factor of activated T cells (NFAT)-dependent transcription was induced by gp120. Transfection of a dominant negative mutant c-Jun protein, TAM-67, efficiently blocked the induction of COX-2 promoter by gp120, confirming AP-1 requirement. Moreover, gp120 rapidly activates the c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase phosphorylation. The importance of NF-kappaB and AP-1 in COX-2 promoter and protein induction was corroborated by using pharmacological NF-kappaB, p38 and JNK inhibitors. PMID: 16001969 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Assay Drug Dev Technol. 2005 Jun;3(3):261-71. Inflammatory pathway analysis using a high content screening platform. Bertelsen M, Sanfridson A. Department of Biological Sciences, AstraZeneca R&D, Lund, Sweden. Malene.Bertelsen@astrazeneca.com High content cellular screening assays are useful tools to investigate the interplay between signaling pathways and offer valuable platforms to determine the mode of action, potency, and selectivity of potential drug candidates in a biological setting. We describe a cell-based multiplex fluorescent imaging assay that permits concurrent detection and quantification of the distribution of nuclear factor kappaB (NFkappaB) p65/RelA and phosphorylated forms of p38 and c-Jun between the cytosol and nucleus. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). A significant window between untreated and interleukin-1alpha (IL-1alpha) stimulated HeLa cells for all three targets was achieved with low variability. Staining specificity was confirmed with blocking peptides and pharmacological inhibition of p38, c-Jun-N-terminal kinase (JNK), and inhibitory kappaB kinase 2, and channel bleed-through was eliminated or counterbalanced by the use of fixed exposure times together with careful reporter channel selection. The JNK inhibitor SP600125 was used as a demonstration compound because in addition to inhibiting nuclear accumulation of phosphorylated c-Jun it reduced nuclear translocation of phosphorylated p38 and NFkappaB p65/RelA in a dose-dependent manner, indicating a lack of SP600125 selectivity. This was supported by RNA interference where co-transfection of small interfering RNA targeting both JNK1 and JNK2, to limit signaling redundancy, significantly inhibited IL-1alpha-stimulated translocation of phosphorylated c-Jun without altering phosphorylated p38 and NFkappaB p65/RelA redistribution. This image analysis application is a valuable and information-rich screening tool to investigate compound selectivity and/or cross-talk between key signaling pathways involved in the inflammatory response. PMID: 15971988 [PubMed - in process] --------------------------------------------------------------- 4: Mol Cell Biol. 2005 Jul;25(13):5429-44. Blockade of histone deacetylase inhibitor-induced RelA/p65 acetylation and NF-kappaB activation potentiates apoptosis in leukemia cells through a process mediated by oxidative damage, XIAP downregulation, and c-Jun N-terminal kinase 1 activation. Dai Y, Rahmani M, Dent P, Grant S. Department of Medicine, Virginia Commonwealth University/Massey Cancer Center, Richmond, Virginia 23298, USA. NF-kappaB activation is reciprocally regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and deacetylases (HDACs). Here we demonstrate that in leukemia cells, NF-kappaB activation by the HDAC inhibitors (HDACIs) MS-275 and suberoylanilide hydroxamic acid was associated with hyperacetylation and nuclear translocation of RelA/p65. The latter events, as well as the association of RelA/p65 with IkappaBalpha, were strikingly diminished by either coadministration of the IkappaBalpha phosphorylation inhibitor Bay 11-7082 (Bay) or transfection with an IkappaBalpha superrepressor. Inhibition of NF-kappaB by pharmacological inhibitors or genetic strategies markedly potentiated apoptosis induced by HDACIs, and this was accompanied by enhanced reactive oxygen species (ROS) generation, downregulation of Mn-superoxide dismutase and XIAP, and c-Jun N-terminal kinase 1 (JNK1) activation. Conversely, N-acetyl L-cysteine blocked apoptosis induced by Bay/HDACIs by abrogating ROS generation. Inhibition of JNK1 activation attenuated Bay/HDACI lethality without affecting NF-kappaB inactivation and ROS generation. Finally, XIAP overexpression dramatically protected cells against the Bay/HDACI regimen but failed to prevent ROS production and JNK1 activation. Together, these data suggest that HDACIs promote the accumulation of acetylated RelA/p65 in the nucleus, leading to NF-kappaB activation. Moreover, interference with these events by either pharmacological or genetic means leads to a dramatic increase in HDACI-mediated lethality through enhanced oxidative damage, downregulation of NF-kappaB-dependent antiapoptotic proteins, and stress-related JNK1 activation. PMID: 15964800 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Clin Cancer Res. 2004 Oct 15;10(20):6821-9. Induction of thyroid cancer cell apoptosis by a novel nuclear factor kappaB inhibitor, dehydroxymethylepoxyquinomicin. Starenki DV, Namba H, Saenko VA, Ohtsuru A, Maeda S, Umezawa K, Yamashita S. Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. PURPOSE: The objective of the study was to determine the effects of a novel selective nuclear factor kappaB (NF-kappaB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in thyroid carcinoma cells in vitro and in vivo and to additionally elucidate the molecular mechanisms underlying the action of this chemotherapeutic agent. EXPERIMENTAL DESIGN: In the in vitro experiments, the induction of apoptosis by DHMEQ in various human thyroid carcinoma cell types was determined by flow cytometry analysis of annexin-V binding and the caspase activation by Western blotting. For the in vivo study, female nu/nu mice were xenografted with s.c. FRO thyroid tumors. DHMEQ solution was injected i.p. at a dose of 8 mg/kg/day for two weeks. Tumor dimensions were monitored twice weekly, and apoptosis in tumor specimens was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. RESULTS: Treatment with DHMEQ substantially inhibited the translocation of p65 and p50 NF-kappaB subunits to the nucleus, the DNA-binding activity of the RelA/p65, NF-kappaB-dependent expression of the inhibitor of apoptosis (IAP)-family proteins, cIAP-1, cIAP-2, and XIAP, and the de novo synthesis of inhibitor of nuclear factor kappaB alpha. At concentration levels ranging from 0.1 to 5 microg/ml, DHMEQ induced a caspase-mediated apoptotic response that could be abrogated by the c-Jun NH(2)-terminal kinase inhibitor SP600125 but not by either mitogen-activated protein/extracellular signal-regulated kinase kinase or p38 inhibitors. In contrast, normal human thyrocytes were resistant to DHMEQ-induced apoptosis. At higher doses of DHMEQ we observed the necrotic-like killing of both normal and malignant thyrocytes, which was resistant to mitogen-activated protein kinase inhibitors. In nude mice DHMEQ substantially inhibited tumor growth without observable side effects, and increased numbers of apoptotic cells were observed in the histologic sections of tumors treated with DHMEQ. CONCLUSIONS: Our results show the potential usefulness of the novel NF-kappaB inhibitor, DHMEQ, in future therapeutic strategies for the treatment of thyroid cancers that do not respond to conventional approaches. PMID: 15501958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Biol Chem. 2004 Dec 31;279(53):55633-43. Epub 2004 Oct 15. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. Buss H, Dorrie A, Schmitz ML, Hoffmann E, Resch K, Kracht M. Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany. Phosphorylation of NF-kappaB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IkappaB kinase (IKK)-alpha/beta (the best characterized Ser-536 kinases so far) or the IKKbeta inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKalpha/beta-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKalpha, IKKbeta, IKKepsilon, and TBK1. Overexpressed IKKepsilon and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter. PMID: 15489227 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Mol Cell Biol. 2004 Nov;24(21):9317-26. Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL. Kwon HJ, Breese EH, Vig-Varga E, Luo Y, Lee Y, Goebl MG, Harrington MA. Department of Biochemistry and Molecular Biology, 635 Barnhill Dr., MS 4071, Indianapolis, IN 46202-5122, USA. A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IkappaB kinase beta (IKK-beta), IKK-alpha, and IKK-gamma/N, leading to changes in NF-kappaB-dependent gene expression. However, it is not clear how the NF-kappaB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-alpha)-dependent but not interleukin-1-dependent NF-kappaB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-kappaB activity. SIMPL interacts with nuclear p65 in a TNF-alpha-dependent manner to promote endogenous NF-kappaB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-alpha activation of NF-kappaB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-alpha-specific induction of gene expression. PMID: 15485901 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Zhonghua Yi Xue Za Zhi. 2004 Aug 2;84(15):1235-9. [The therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine in mice with acute necrotizing pancreatitis] [Article in Chinese] Li L, Wang XP, Wu K. Department Of Gastroentorology, Shanghai First Hospital, Shanghai Jiaotong University, 200080 Shanghai, China. OBJECTIVE: To investigate the therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OXPAPC) in animal model of acute necrotizing pancreatitis (ANP) and its possible mechanism. METHODS: Forty-eight C57BL/6J mice were randomly divided into two groups, ANP group and ANP treated with OXPAPC group. ANP model was induced by seven times administration of cerulein (50 microg/kg) challenged by lipopolysaccharide (LPS) (5 mg/kg) intraperitoneal injection. Treatment with OXPAPC was started 5 mins before LPS injection. At the 9th hour, 12nd hour and 24th hour after the first injection of cerulein, serum levels of amylase and lactate dehydrogenase (LDH) were measured. Severity of pancreatitis was evaluated by histological scoring system. The activity of myeloperoxidase (MPO) in pancreas was determined by zymohistochemistry. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA expressions in pancreas were studied by semi-quantitative RT-PCR. The activation of nuclear factor kappaB (NF-kappaB) and c-Jun-N-terminal kinase 1 (JNK1) were investigated by the methods of western blot. RESULTS: Treatment with OXPAPC significantly decreased serum amylase and LDH levels at the 9th hr and 12nd hour. Histologically, OXPAPC reduced the severity of pancreatic injury including inflammatory cell infiltration and necrosis at 12nd hour and 24th hour. There was a significant decrease of MPO activity in OXPAPC group as compared to ANP group. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA levels were reduced in OXPAPC-treated group. Western blot showed down-regulation of NF-KappaB p65 and JNK1 expression were found in OXPAPC-treated mice. CONCLUSION: OXPAPC decreased the severity of experimental acute pancreatitis in mice and inhibited the intrapancreatic inflammatory mediators expression in vivo. The protective effect of OXPAPC was mediated, at least in part, through blocking endotoxin signal pathway. PMID: 15387956 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Int J Oncol. 2004 Oct;25(4):1081-7. Modulation of the transactivation function of nuclear factor-kappaB by lipopolysaccharide in RAW264.7 macrophages. Jung SW, Lee MH, Kim YC, Paik SG, Choi YH, Kim YS, Kang KI. Institute of Biotechnology, Chungnam National University, Daejeon 305-764, Korea. In macrophages, nuclear factor kappa B (NF-kappaB) activation has important roles for the regulation of an inducible nitric oxide synthase (iNOS), several pro-inflammatory cytokines, and anti-apoptotic proteins. In order to analyze the transactivation process of NF-kappaB by lipopolysaccharide (LPS), we used the GAL4-NF-kappaB-p65 fusion protein. This chimeric NF-kappaB is activated transcriptionally only if NF-kappaB transactivation domain is active. With this system, we found that LPS can enhance the transactivation of GAL-NF-kappaB-p65 subunit independent of DNA binding ability and inhibitor of kappaB (IkappaB) regulation. Interestingly, this transactivation by LPS was eliminated with the treatment of U0126, specific inhibitor of mitogen-activated protein kinase (MAPK) kinases (MEKs) 1/2 which has little effect on NF-kappaB activation. We also investigated the effect of inhibitors of apoptosis (IAPs), which might be involved in LPS responses and c-Jun amino terminal kinase (JNKs) activation, on the transactivation of GAL-NF-kappaB-p65. The cIAP1, cIAP2 and XIAP could enhance the NF-kappaB transcription and the chimeric NF-kappaB-p65 transactivation. However, survivin decreased the NF-kappaB transcription and did not influence significantly the chimeric NF-kappaB-p65 transactivation. Taken together, LPS-dependent NF-kappaB transactivation may be involved in extracellular signal-regulated kinase (ERK) pathway and IAPs. PMID: 15375559 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Blood. 2004 Nov 15;104(10):3302-4. Epub 2004 Jul 27. Comparative analysis of T-cell costimulation and CD43 activation reveals novel signaling pathways and target genes. Mattioli I, Dittrich-Breiholz O, Livingstone M, Kracht M, Schmitz ML. Department of Chemistry and Biochemistry, University of Bern, Freiestr. 3, 3012 Bern, Switzerland. The CD43 lymphocyte surface receptor is involved in the regulation of lymphocyte adhesion and activation. Many CD43 functions remain controversial or unclear, and it is not known to which extent CD43 signaling pathways are shared with or distinct from those used by the T-cell receptor (TCR). Here, we systematically compared signaling events and target gene expression induced by CD43 or T-cell costimulation in primary human peripheral T cells. These studies identify nuclear factor-kappaB (NF-kappaB) p65 serine 468 as a novel inducible phosphorylation site strongly induced by T-cell costimulation and only weakly triggered by CD43 ligation. We also identified CD43 as a novel Jun N-terminal kinase (JNK) activator and a comprehensive analysis of further signaling events suggests that both stimuli use overlapping but also distinct signaling pathways. Microarray analysis of inflammatory genes shows 1 group of genes coregulated by both stimuli and 2 further groups of target genes affected solely by costimulation or primarily by CD43. PMID: 15280197 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Biol Chem. 2004 Sep 17;279(38):39541-54. Epub 2004 Jul 13. Genetic deletion of glycogen synthase kinase-3beta abrogates activation of IkappaBalpha kinase, JNK, Akt, and p44/p42 MAPK but potentiates apoptosis induced by tumor necrosis factor. Takada Y, Fang X, Jamaluddin MS, Boyd DD, Aggarwal BB. Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. Glycogen synthase kinase (GSK)-3beta is a constitutively active, proline-directed serine/threonine kinase that controls growth modulation and tumorigenesis through multiple intracellular signaling pathways. How GSK-3beta regulates signaling pathways induced by cytokines such as tumor necrosis factor (TNF) is poorly understood. In this study, we used fibroblasts derived from GSK-3beta gene-deleted mice to understand the role of this kinase in TNF signaling. TNF induced NF-kappaB activation as measured by DNA binding in wild-type mouse embryonic fibroblasts, but deletion of GSK-3beta abolished this activation. This inhibition was due to suppression of IkappaBalpha kinase activation and IkappaBalpha phosphorylation, ubiquitination, and degradation. TNF-induced NF-kappaB reporter gene transcription was also suppressed in GSK-3beta gene-deleted cells. NF-kappaB activation induced by lipopolysaccharide, interleukin-1beta, or cigarette smoke condensate was completely suppressed in GSK-3beta(-/-) cells. Deletion of GSK-3beta also abolished TNF-induced c-Jun N-terminal kinase and p44/p42 mitogen-activated kinase activation. Most surprisingly, TNF-induced Akt activation also required the presence of GSK-3beta. TNF induced expression of the NF-kappaB-regulated gene products cyclin D1, COX-2, MMP-9, survivin, IAP 1, IAP 2, Bcl-x(L), Bfl-1/A1, TRAF1, and FLIP in wild-type mouse embryonic fibroblasts but not in GSK-3beta(-/-) cells, and this correlated with potentiation of TNF-induced apoptosis as indicated by cell viability, annexin V staining, and caspase activation. Overall, our results indicate that GSK-3beta plays a critical role in TNF signaling and in the signaling of other inflammatory stimuli and that its suppression can be exploited as a potential target to inhibit angiogenesis, proliferation, and survival of tumor cells. PMID: 15252041 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Mol Pharmacol. 2004 Oct;66(4):956-63. Epub 2004 Jul 2. Contribution of disruption of the nuclear factor-kappaB pathway to induction of apoptosis in human leukemia cells by histone deacetylase inhibitors and flavopiridol. Gao N, Dai Y, Rahmani M, Dent P, Grant S. Department of Medicine, Virginia Commonwealth University/Medical College of Virginia, Richmond 23298, USA. Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaB) and suberoylanilide hydroxamic acid (SAHA) have been examined in human leukemia cells in relation to effects on nuclear factor kappaB (NF-kappaB) activation. Exposure (24 h) of U937 human leukemia cells to NaB (1 mM) or SAHA (1.5 microM) resulted in a marked increase in NF-kappaB DNA binding, effects that were essentially abrogated by coadministration of flavopiridol (100 nM). These events were accompanied by a marked increase in mitochondrial injury, caspase activation, and apoptosis. Mutant cells expressing an IkappaBalpha super-repressor exhibited impairment of NF-kappaB DNA binding in response to HDACIs and a significant although modest increase in apoptosis. However, disruption of the NF-kappaB pathway also increased mitochondrial injury and caspase activation in response to flavopiridol and to an even greater extent to the combination of flavopiridol and HDACIs. Coadministration of flavopiridol with HDACIs down-regulated the X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p21CIP1/WAF1 and activated c-Jun NH2-terminal kinase; moreover, these effects were considerably more pronounced in IkappaBalpha mutants. Similar responses were observed in U937 mutant cells stably expressing RelA/p65 small interfering RNA. In all cases, flavopiridol was significantly more potent than genetic interruption of the NF-kappaB cascade in promoting HDACI-mediated lethality. Together, these findings are consistent with the notion that although inhibition of NF-kappaB activation by flavopiridol contributes to antileukemic interactions with HDACIs, other NF-kappaB-independent flavopiridol actions (e.g., down-regulation of Mcl-1, XIAP, and p21CIP1/WAF1) play particularly critical roles in this phenomenon. PMID: 15235103 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Biochim Biophys Acta. 2004 May 28;1692(1):17-24. Activation of the Interleukin-6 promoter by a dominant negative mutant of c-Jun. Faggioli L, Costanzo C, Donadelli M, Palmieri M. Department of Neurological and Vision Sciences, Biochemistry Section, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. The human IL-6 promoter contains multiple regulatory elements such as those binding transcription factors belonging to the NF-kappaB (-75/-63), C/EBP (-158/-145 and -87/-76) and AP-1 (-283/-277) families. Herein, we report that ectopic expression of c-Jun, C/EBPdelta, and the p65 subunit of NF-kappaB synergistically activates an IL-6 promoter construct containing only a TATA box and a kappaB binding site. These results suggest that interactions among NF-kappaB, C/EBP, and AP-1, which are all activated by the most powerful physiological inducers of the IL-6 gene, namely TNF-alpha and IL-1, may be crucial for maximal activation of the IL-6 promoter in response to the two cytokines. Furthermore, we show that a mutated form of c-Jun lacking the transactivation domain (TAM-67) was a much stronger activator of the IL-6 promoter than c-Jun. In combination with p65 and/or C/EBPdelta, TAM-67 also synergistically activated the IL-6 promoter, while it inhibited TNF-alpha induced AP-1 activity directing an AP-1-responsive reporter plasmid. Lastly, electrophoretic mobility shift assay (EMSA) results strongly suggest the formation of complexes between p65, C/EBPdelta, and/or c-Jun or TAM-67 on the kappaB site, supporting the idea that the functional synergism is determined by a physical interaction. These data provide new insight into the molecular mechanisms regulating the formation of the transcription complex responsible for IL-6 promoter activation. PMID: 15158360 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: FEBS Lett. 2004 May 21;566(1-3):183-9. NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway. Je JH, Lee JY, Jung KJ, Sung B, Go EK, Yu BP, Chung HY. Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea. 4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-kappaB) activation in endothelial cells. HHE induced NF-kappaB activation by inhibitor of kappaB (IkappaB) phosphorylation via the IkappaB kinase (IKK)/NF-kappaB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH(2)-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-kappaB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IkappaB phosphorylation, and NF-kappaB luciferase activity. These findings strongly suggest that HHE induces NF-kappaB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells. PMID: 15147892 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: 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] --------------------------------------------------------------- 16: Oncogene. 2004 Jun 10;23(27):4673-80. JNK activation is critical for Aplidin-induced apoptosis. Cuadrado A, Gonzalez L, Suarez Y, Martinez T, Munoz A. Pharma Mar SA, E-28770 Colmenar Viejo, Madrid, Spain. Aplidin is an antitumor drug that induces apoptosis and activates EGFR, Src, JNK and p38MAPK. Here, we show that Aplidin induces c-JUN, JUN B, JUN D, c-FOS, FRA-1 and FOS B genes of the activator-protein (AP)-1 family, and also p65/RELA, a major component of nuclear factor-kappa B (NF-kappaB). Concordantly, Aplidin increases AP-1 and NF-kappaB activity. c-FOS induction depends on EGFR, Src and JNK/p38MAPK. In contrast, induction of c-JUN does not require EGFR activity and p65/RELA induction is only partially dependent on these kinases. We used several genetically deficient cells to identify the critical target of Aplidin. Mouse embryo fibroblasts (MEFs) deficient for src, yes and fyn, and those lacking all p38MAPK isoforms displayed normal Aplidin sensitivity (IC50=12 nM). In contrast, MEFs lacking jnk1 and jnk2, which do not express any JNK isoform, were much less sensitive (IC50>500 nM). Furthermore, cells lacking c-jun or expressing a c-Jun protein in which JNK targets Ser(63/73) were mutated (c-JunAA) showed intermediate sensitivity (IC50=60 nM). Additionally, Aplidin has higher cytotoxic activity against proliferating than quiescent cells, which is reflected in higher JNK activation. We conclude that phosphorylation by JNK of c-Jun and additional substrate(s) is crucial for Aplidin activity. PMID: 15122339 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: J Invest Dermatol. 2004 Apr;122(4):863-72. Histamine enhances the production of granulocyte-macrophage colony-stimulating factor via protein kinase Calpha and extracellular signal-regulated kinase in human keratinocytes. Kanda N, Watanabe S. Department of Dermatology, Teikyo University, School of Medicine, Tokyo, Japan. nmk@med.teikyo-u.ac.jp The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes is related to the chronicity of atopic dermatitis. Mast cell-derived histamine contributes to the cross-talk between mast cells and keratinocytes. We examined the effects of histamine on GM-CSF production in human keratinocytes. Histamine increased GM-CSF secretion, mRNA stability and promoter activity. Activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) elements on the promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of AP-1 and NF-kappaB. Histamine shifted AP-1 composition form c-Jun homodimers to c-Fos/c-Jun heterodimers, and transiently expressed c-Fos protein. Histamine rapidly induced the phosphorylation and degradation of inhibitory kappaB. Histamine induced membrane translocation of protein kinase Calpha. Histamine-induced GM-CSF production was completely abolished by H1 antagonist pyrilamine and conventional protein kinase C inhibitor Go6976, and partially suppressed by PD98059 which inhibits the activation of extracellular signal-regulated kinase. Go6976 and PD98059 suppressed histamine-induced c-Fos expression and AP-1 activation. Go6976 and PD98059 suppressed histamine-induced enhancement of NF-kappaB transcriptional activity. Histamine-induced phosphorylation and degradation of inhibitory kappaB was suppressed by Go6976, but not by PD98059. These results suggest that histamine may enhance GM-CSF production at transcriptional and posttranscriptional levels via H1 receptor, protein kinase Calpha and extracellular signal-regulated kinase. PMID: 15102074 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Eur J Immunol. 2004 Apr;34(4):1196-207. Toll-like receptor stimulation induces airway hyper-responsiveness to bradykinin, an effect mediated by JNK and NF-kappa B signaling pathways. Bachar O, Adner M, Uddman R, Cardell LO. Department of Otorhinolaryngology, Malmo University Hospital, Malmo, Sweden. Ofir.Bachar@oron.mas.lu.se Airway infections induce hyper-responsiveness in asthmatic patients. Toll-like receptors (TLR) mediate inflammatory responses to microbes. Occurrence and effects of TLR2, TLR3 and TLR4 were examined in a mouse organ culture model of asthma focusing on the smooth muscle responses to bradykinin. TLR2, TLR3 and TLR4 mRNA, and TLR2 and TLR4 immunoreactivity were detected in the tracheal muscle layer. Tracheal organ culture for 1 or 4 days with lipopolysaccharide (LPS; TLR2/4 agonist) or polyinosinic polycytidylic acid (poly-I-C; TLR3 agonist) enhanced bradykinin- and [des-Arg(9)]-bradykinin-induced contractions. Simultaneous LPS and poly-I-C treatment resulted in synergistic enhancement of bradykinin-induced contraction. In carbachol-pre-contracted segments TLR stimulation induced less potent relaxations to bradykinin and [des-Arg(9)]-bradykinin. The LPS and poly-I-C enhancement of bradykinin-induced contraction was inhibited by the transcriptional inhibitor actinomycin-D, dexamethasone, the proteasome inhibitor MG-132 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125. LPS and poly-I-C induced translocation of NF-kappa B p65 to the nucleus and up-regulation of kinin B(1) and B(2) receptor mRNA. In summary, TLR2, TLR3 and TLR4 are expressed in the mouse tracheal smooth muscle. Costimulation of these receptors results in NF-kappa B- and JNK-mediated transcription of B(1) and B(2) receptor, inducing hyper-responsiveness to bradykinin. PMID: 15048731 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Cancer Res. 2004 Mar 15;64(6):1902-5. Reactive oxygen species (ROS), troublemakers between nuclear factor-kappaB (NF-kappaB) and c-Jun NH(2)-terminal kinase (JNK). Zhang Y, Chen F. Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai, P. R. China. Nuclear factor-kappaB (NF-kappaB) and c-Jun NH(2)-terminal kinase (JNK) are activated simultaneously under a variety of stress conditions. They also share several common signaling pathways for their activation in response to cytokines or growth factors. Recent studies, however, demonstrated a new form of interplay between these two allies. Inhibition of NF-kappaB by ikkbeta or rela gene deficiency sensitizes stress responses through enhanced or prolonged activation of JNK. Conversely, sustained activation of NF-kappaB inhibits cytokine-induced JNK activation. The mechanisms of how NF-kappaB and JNK become rivals for each other are under extensive debate. Publication Types: Review Review, Tutorial PMID: 15026320 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Eur J Biochem. 2004 Mar;271(6):1094-105. NF-kappaB- and c-Jun-dependent regulation of human cytomegalovirus immediate-early gene enhancer/promoter in response to lipopolysaccharide and bacterial CpG-oligodeoxynucleotides in macrophage cell line RAW 264.7. Lee Y, Sohn WJ, Kim DS, Kwon HJ. Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejon, Korea. The cytomegalovirus immediate-early (CMV IE) gene enhancer/promoter regulates the expression of immediate-early gene products and initiation of CMV replication. TNF-alpha and lipopolysaccharide (LPS) strongly activate the promoter, possibly involving NF-kappaB. CpG-oligodeoxynucleotides (CpG-ODNs), which contain unmethylated CpG dinucleotides in the context of particular base sequences, have gained attention because of their stimulating effects, via NF-kappaB, which have a strong innate immune response. To study the effects of LPS and CpG-ODNs, as well as the mechanisms of their actions regarding CMV IE enhancer/promoter activation, we used a macrophage cell line, RAW 264.7. Stimulation of the cells with LPS or CpG-ODNs resulted in the activation of the CMV IE enhancer/promoter. We examined the involvement of NF-kappaB and c-Jun transcription factors by promoter deletion/site-specific mutation analysis and ectopic expression, and found them to have additive effects. Involvement of myeloid differentiation protein, an upstream regulator of NF-kappaB and c-Jun, was also investigated. Experimental results indicate that both LPS-induced and CpG-ODN-induced activations of CMV IE enhancer/promoter are mediated by Toll-like receptor signaling molecules. Several lines of evidence suggest the potential contribution of bacterial infection in CMV reactivation along with the potential application of CpG-ODNs in gene therapy as a stimulator for the optimal expression of target genes under the control of the CMV IE enhancer/promoter. PMID: 15009188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Immunol. 2004 Feb 15;172(4):2341-51. Immunosuppressive activity of endovanilloids: N-arachidonoyl-dopamine inhibits activation of the NF-kappa B, NFAT, and activator protein 1 signaling pathways. Sancho R, Macho A, de La Vega L, Calzado MA, Fiebich BL, Appendino G, Munoz E. Departamento de Biologia Celular, Fisiologia e Inmunologia, Universidad de Cordoba, Facultad de Medicina, Cordoba, Spain. Endogenous N-acyl dopamines such as N-arachidonoyldopamine (NADA) and N-oleoyldopamine have been recently identified as a new class of brain neurotransmitters sharing endocannabinoid and endovanilloid biological activities. As endocannabinoids show immunomodulatory activity, and T cells play a key role in the onset of several diseases that affect the CNS, we have evaluated the immunosuppressive activity of NADA and N-oleoyldopamine in human T cells, discovering that both compounds are potent inhibitors of early and late events in TCR-mediated T cell activation. Moreover, we found that NADA specifically inhibited both IL-2 and TNF-alpha gene transcription in stimulated Jurkat T cells. To further characterize the inhibitory mechanisms of NADA at the transcriptional level, we examined the DNA binding and transcriptional activities of NF-kappaB, NF-AT, and AP-1 transcription factors in Jurkat cells. We found that NADA inhibited NF-kappaB-dependent transcriptional activity without affecting either degradation of the cytoplasmic NF-kappaB inhibitory protein, IkappaBalpha, or DNA binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by NADA in stimulated cells. In addition, NADA inhibited both binding to DNA and the transcriptional activity of NF-AT and AP-1, as expected from the inhibition of NF-AT1 dephosphorylation and c-Jun N-terminal kinase activation in stimulated T cells. Finally, overexpression of a constitutively active form of calcineurin demonstrated that this phosphatase may represent one of the main targets of NADA. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA and highlight their potential to design novel therapeutic strategies to manage inflammatory diseases. PMID: 14764703 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: J Leukoc Biol. 2004 Apr;75(4):689-97. Epub 2004 Jan 23. Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-kappaB-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding. Darieva Z, Lasunskaia EB, Campos MN, Kipnis TL, Da Silva WD. Laboratoire de Biologia do Reconhecer, Universisade Estadual do Norte Fluminense, Campos, Rio de Janeiro, Brazil. The proinflammatory response of infected macrophages is an important early host defense mechanism against mycobacterial infection. Mycobacteria have been demonstrated to induce proinflammatory gene transcription through the Toll-like receptors, (TLR)2 and TLR 4, which initiate signaling cascades leading to nuclear factor (NF)-kappaB activation. The main transduction pathway responsible for NF-kappaB activation has been established and involves the MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor-6, NF-kappaB-inducing kinase, and inhibitor of kappaB kinase complex. The role of other kinase cascades triggered by mycobacteria in the NF-kappaB activation is less clear. We herein examine the role of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascades in the expression of the bacillus Calmette-Guerin (BCG) mycobacteria-induced NF-kappaB-dependent genes, macrophage-inflammatory protein-2 (MIP-2) and inducible nitric oxide (NO) synthase. Specific pharmacological inhibition of the PI-3K, c-jun-N-terminal kinase (JNK), and to a smaller extent, p38 MAPK but not extracellular-regulated kinase (ERK), suppressed NF-kappaB-dependent reporter gene transcription and MIP-2 and NO secretion in BCG-induced RAW264.7 macrophages. A similar effect was obtained following molecular inhibition of JNK via JNK-interacting protein-1 overexpression. In addition, a kinase-dead mutant of MEK kinase-1, the up-stream regulator of JNK, also proved to be a potent inhibitor of NF-kappaB-reporter activity. The effect of inhibitors was mediated by the down-regulation of NF-kappaB transcription activity and without effecting its nuclear translocation. These data suggest an indirect mechanism of the NF-kappaB regulation by these kinases, probably through p65 phosphorylation and improved binding to the p300 transcription coactivator. The data obtained demonstrate that PI-3K, JNK, and p38 MAPK activation by mycobacteria enhance NF-kappaB-driven gene expression contributing to the proinflammatory macrophage response. PMID: 14742634 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: 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] --------------------------------------------------------------- 24: Cancer Res. 2003 Nov 15;63(22):7689-93. Inhibitor of nuclear factor kappaB kinase deficiency enhances oxidative stress and prolongs c-Jun NH2-terminal kinase activation induced by arsenic. Chen F, Castranova V, Li Z, Karin M, Shi X. Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505, USA. lfd3@cdc.gov Stress signals activate both inhibitor of nuclear factor-kappaB kinase (IKKbeta) and c-Jun NH(2)-terminal kinase (JNK). It was shown recently that IKK-dependent nuclear factor kappaB activation results in attenuation of tumor necrosis factor alpha-induced JNK activation. How that negative cross-talk between nuclear factor kappaB and JNK occurs is not well-understood. By using wild-type and Ikkbeta gene knockout (Ikkbeta(-/-)) mouse embryo fibroblasts, we found that IKKbeta deficiency results in prolongation of arsenic-induced JNK activation, which was not due to the decreased expression of GADD45beta or X-linked Inhibitor of Apoptosis (XIAP), as suggested previously for RelA(-/-) cells treated with tumor necrosis factor alpha. This enhanced JNK activation was largely associated with an oxidative stress response as indicated by elevated expression of heme oxygenase-1 and the accumulation of H(2)O(2) in Ikkbeta(-/-) cells. Expression profiling experiments revealed an increased expression of p450 family CYP1B1 mRNA in Ikkbeta(-/-) cells compared with wild-type cells. Inhibition of CYP1B1 reduced both oxidative stress and arsenic-stimulated JNK activation. Thus, increased CYP1B1 expression is central to and seems to be responsible for sensitizing Ikkbeta(-/-) cells to stress-induced JNK activation. PMID: 14633691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Cancer Res. 2003 Nov 1;63(21):7106-12. Interruption of nuclear factor kappaB signaling by the androgen receptor facilitates 12-O-tetradecanoylphorbolacetate-induced apoptosis in androgen-sensitive prostate cancer LNCaP cells. Altuwaijri S, Lin HK, Chuang KH, Lin WJ, Yeh S, Hanchett LA, Rahman MM, Kang HY, Tsai MY, Zhang Y, Yang L, Chang C. George Whipple Laboratory for Cancer Research, Department of Pathology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA. 12-O-tetradecanoylphorbolacetate (TPA) influences proliferation, differentiation, and apoptosis in a variety of cells including prostate cancer cells. Here, we show that androgen treatment potentiates TPA-induced apoptosis in androgen-sensitive prostate cancer LNCaP cells but not in androgen-independent prostate cancer cell lines DU145 and PC-3. The use of the antiandrogen bicalutamide (Casodex) rescued LNCaP cells from 5-alpha-dihydrotestosterone (DHT)/TPA-induced apoptosis, suggesting that DHT/TPA-induced apoptosis is mediated by androgen/androgen receptor (AR). In addition, a caspase-3 inhibitor (Ac-DEVD-CHO) reduced the level of apoptosis, suggesting that DHT/TPA-mediated apoptosis occurs through a caspase-3-dependent pathway. A functional reporter assay using nuclear factor (NF) kappaB-luciferase and an electromobility gel shift assay showed that DHT suppressed NFkappaB activity. In addition, apoptosis mediated by combined DHT/TPA treatment was abrogated by overexpression of the NFkappaB subunit p65 in LNCaP-p65 cells, suggesting that NFkappaB may play an important role in regulating the effects of androgen/AR and TPA on apoptosis. Furthermore, use of the c-Jun N-terminal kinase (JNK) inhibitor SB202190 showed that the combination of DHT/TPA increased JNK activation in LNCaP cells but not in LNCaP-p65 cells, demonstrating that NFkappaB may be able to suppress JNK activity. These results indicate that androgen/AR facilitates TPA-induced apoptosis by interruption of the NFkappaB signaling pathway, leading to activation of JNK in LNCaP cells. These data describe a signaling pathway that could potentially be useful in proposed therapeutic treatment strategies exploiting combinations of different agents that control apoptosis in prostate tumors. PMID: 14612503 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Biochem Biophys Res Commun. 2003 Aug 29;308(3):608-13. Regulation of macrophage inflammatory protein-2 gene expression in response to oligodeoxynucleotide containing CpG motifs in RAW 264.7 cells. Kwon HJ, Kim DS. Institute of Life Science and Biotechnology, College of Science, Yonsei University, Seoul, South Korea. Macrophage inflammatory protein-2 (MIP-2) is a mouse C-X-C chemokine that plays an important role in the recruitment of neutrophils. The unregulated production of MIP-2 has been associated with inflammatory diseases such as arthritis, glomerulonephritis, and sepsis. We have shown that the MIP-2 gene expression is transcriptionally activated by synthetic oligodeoxynucleotide (ODN) containing unmethylated CpG dinucleotides in the context of particular base sequences (CpG-ODN) in a CpG sequence-dependent manner. Inhibition of NF-kappaB nuclear localization by coexpression of a mutant IkappaBalpha protein blocked CpG-ODN-induced transcription from a MIP-2 promoter-reporter construct, showing that NF-kappaB activation is required for MIP-2 gene expression in the CpG-ODN-signaling pathway. We also provided evidence that NF-kappaB and c-Jun contributes to the expression of MIP-2 gene in response to CpG-ODN, since ectopical expression of NF-kappaB and c-Jun in RAW 246.7 cells leads to dramatically increase the ability of CpG-ODN 1826(S) in MIP-2 promoter activity. These results perhaps give more insights into understanding of the mechanisms involved in transient inflammatory arthritis induction by CpG-ODN treatment. PMID: 12914794 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Cancer Res. 2003 May 1;63(9):2206-15. Constitutive activation of nuclear factor kappaB p50/p65 and Fra-1 and JunD is essential for deregulated interleukin 6 expression in prostate cancer. Zerbini LF, Wang Y, Cho JY, Libermann TA. BIDMC Genomics Center and New England Baptist Bone and Joint Institute, Boston, Massachusetts 02115, USA. To date, no effective treatment for patients with advanced androgen-independent prostate cancer is available, whereas androgen ablation therapy, surgery, and radiation therapy are effective in treating local, androgen-dependent tumors. The mechanisms underlying the differences between androgen-dependent and -independent prostate cancer remain elusive. Interleukin (IL)-6 is a pleiotropic cytokine whose expression under normal physiological conditions is tightly controlled. However, aberrant constitutive IL-6 gene expression has been implicated in prostate cancer progression and resistance to chemotherapy and has been directly linked to prostate cancer morbidity and mortality. Particularly striking is the large increase in the expression of IL-6 in hormone-refractory prostate cancer. IL-6, in addition to its role as an immunomodulatory cytokine, functions as a growth and differentiation factor for prostate cancer cells. To determine the molecular mechanisms that lead to deregulated IL-6 expression in advanced prostate cancer, we examined the regulatory elements involved in IL-6 gene expression in androgen-independent prostate cancer cells. We demonstrate that, in contrast to the androgen-sensitive LNCaP cells, androgen-insensitive PC-3 and DU145 cells express high levels of IL-6 protein and mRNA due to enhanced promoter activity. Deregulated activation of the IL-6 promoter is for the most part mediated by a combined constitutive activation of the nuclear factor (NF)-kappaB p50 and p65 and the activator protein 1 (AP-1) JunD and Fra-1 family members as demonstrated by electrophoretic mobility shift assays, site-directed mutagenesis, and transfection experiments. Mutation of the NF-kappaB and AP-1 sites drastically reduces IL-6 promoter activity in both androgen-independent prostate cancer cell lines. Additionally, inhibition of these transcription factors using adenovirus vectors encoding either the IkappaBalpha repressor gene or a dominant negative JunD mutant leads to a strong down-regulation of IL-6 gene expression at the mRNA and protein level as measured by real-time PCR and ELISA, respectively. Furthermore, the blockade of IL-6 gene expression results in drastic inhibition of the constitutively activated signal transducers and activators of transcription 3 signaling pathway in DU145 cells. Our data demonstrate for the first time that a combined aberrant activation of NF-kappaB p50 and p65 and AP-1 JunD and Fra-1 in androgen-independent prostate cancer cells results in deregulated IL-6 expression, suggesting a novel potential entry point for therapeutic intervention in prostate cancer. PMID: 12727841 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: FASEB J. 2003 Jun;17(9):1099-101. Epub 2003 Apr 8. Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IkappaB-alpha kinase inhibition and cell death induction. Zernecke A, Erl W, Fraemohs L, Lietz M, Weber C. Institut fur Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-Universitat Munchen, 80336 Munchen, Germany. The cyclopentenone prostaglandins (cPG) 15-deoxy-Delta12,14-prostaglandin J2 (dPGJ2) and PGA1 can inhibit multiple steps in nuclear factor (NF)-kappaB signaling and can induce cell death. Here we characterized the effects of dPGJ2 and PGA1 on the inflammatory induction of endothelial cell adhesion molecules (CAM). Pretreatment of endothelial cells with dPGJ2 or PGA1 at low concentrations dose dependently inhibited the up-regulation of CAM expression and monocyte arrest by tumor necrosis factor (TNF)-alpha but not expression of inhibitor of apoptosis proteins. Only at high concentrations, cPG enhanced TNF-alpha-induced cell death and inhibited TNF-alpha-induced IkappaB-alpha kinase (IKK) activation, IkappaB-alpha degradation, and NF-kappaB/p65 translocation, while promoting AP-1/c-jun phosphorylation. Expression of an IKK-beta mutant (C179A) resistant to interaction with cPG impaired cell death induction but not inhibition of CAM up-regulation by cPG. Gel shift and reporter gene analysis revealed that cPG at low concentrations directly impaired DNA binding of NF-kappaB and NF-kappaB-dependent transactivation. The synthetic analogs dPGA1 or dPGA2 were ineffective, indicating structural specificity of cPG. Thus, the suppression of endothelial CAM up-regulation with cPG is dissociated from cell death sensitization and IKK inhibition above threshold concentrations and related to interference with NF-kappaB binding. Our findings define distinct mechanisms for anti-inflammatory and proapoptotic effects of cPG in endothelial cells. PMID: 12692090 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Biol Cell. 2003 Apr;14(4):1334-45. Erratum in: Mol Biol Cell.2003 May;14(5):following table of contents. Choudhury JA [corrected to Ahmed-Choudhury J]. Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or intrahepatic endothelial cell proliferation. Ahmed-Choudhury J, Russell CL, Randhawa S, Young LS, Adams DH, Afford SC. Liver Research Laboratories, Medical Research Council Centre for Immune Regulation, University of Birmingham Institute of Clinical Science, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, United Kingdom. CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-kappaB activity in hepatocytes,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-kappaB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase 1/2) and expression of inhibitor kappaBalpha were entirely consistent, and thus confirmed the profiles of NF-kappaB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-kappaB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by approximately 50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-kappaB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-kappaB activation and a lack of AP-1 activation in IHECs result in proliferation. PMID: 12686591 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Immunol. 2003 Jan 1;170(1):508-19. Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components. Dobrovolskaia MA, Medvedev AE, Thomas KE, Cuesta N, Toshchakov V, Ren T, Cody MJ, Michalek SM, Rice NR, Vogel SN. Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201, USA. In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway. PMID: 12496438 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Mol Carcinog. 2002 Dec;35(4):157-62. Mechanism of action of a dominant negative c-jun mutant in inhibiting activator protein-1 activation. Thompson EJ, Gupta A, Stratton MS, Bowden GT. Department of Pharmacology and Toxicology, The University of Arizona, Tucson, USA. The dominant negative c-jun TAM-67 has been shown to inhibit tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid (OA). To better understand this phenomenon, we investigated the mechanism of action of TAM-67 in response to OA. To identify the mechanism of action, we used a 6xHis-tagged TAM-67 as well as chimeric constructs of TAM-67 that either cannot bind DNA or cannot heterodimerize with wild-type transcription factors. The results of these studies indicated that TAM-67 acts by blocking or squelching. The results of elecrophoretic mobility-shift assays showed that TAM-67 must act by squelching in response to OA, as TAM-67 cannot be found in DNA-binding complexes. We then identified some of the proteins with which TAM-67 interacts. They include all members of the jun and fos families as well as the cAMP response element binding protein, activating transcription factor-1, activating transcription factor-2, and RelA (p65). Thus, we have shown that TAM-67 squelches the induction of activating transcription factor-1 transactivation in response to OA and that TAM-67 is capable of interacting with proteins that control transactivation by binding to the 12-O-tetradecanoylphorbol-13-acetate response element, cAMP response element and nuclear factor-kappaB sites. Copyright 2002 Wiley-Liss, Inc. PMID: 12489106 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Immunol. 2002 Dec 15;169(12):7026-38. Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signal-regulated kinase- and cyclic adenosine 5'-monophosphate (cAMP)-dependent pathways: involvement of NF-kappa B, activator protein 1, and cAMP response element binding protein. Jaramillo M, Olivier M. Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Quebec, Pavillon du Centre Hospitalier de l'Universite Laval, and Departement de Biologie Medicale, Faculte de Medecine, Universite Laval, Ste-Foy, Quebec, Canada. Hydrogen peroxide (H(2)O(2)) has been shown to act as a second messenger that activates chemokine expression. In the present study, we investigated the mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that H(2)O(2) increases mRNA expression of various chemokines, macrophage-inflammatory protein (MIP)-1alpha/CC chemokine ligand (CCL)3, MIP-1beta/CCL4, MIP-2/CXC chemokine ligand 2, and monocyte chemoattractant protein-1/CCL2, by activating the extracellular signal-regulated kinase (ERK) pathway and the nuclear translocation of the transcription factors NF-kappaB, AP-1, and CREB. Blockage of the ERK pathway with specific inhibitors against mitogen-activated protein kinase kinase 1/2 and ERK1/ERK2 completely abolished both the H(2)O(2)-mediated chemokine up-regulation and the activation of all NF studied. Similarly, selective inhibition of cAMP and NF-kappaB strongly down-regulated the induction of all chemokine transcripts as well as CREB and NF-kappaB activation, respectively. Of interest, we detected a significant decrease of NF-kappaB, AP-1, and CREB DNA binding activities by reciprocal competition for these binding sites when either specific cold oligonucleotides (NF-kappaB, AP-1, and CREB) or Abs against various transcription factor subunits (p50, p65, c-Fos, Jun B, c-Jun, and CREB-1) were added. These findings indicate that cooperation between ERK- and cAMP-dependent pathways seems to be required to achieve the formation of an essential transcriptional factor complex for maximal H(2)O(2)-dependent chemokine modulation. Finally, experiments performed with actinomycin D suggest that H(2)O(2)-mediated MIP-1beta mRNA up-regulation results from transcriptional control, whereas that of MIP-1alpha, MIP-2, and monocyte chemoattractant protein-1 is due to both gene transcription activation and mRNA posttranscriptional stabilization. PMID: 12471138 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Mol Cell Biol. 2002 Dec;22(23):8175-83. The p65/RelA subunit of NF-kappaB suppresses the sustained, antiapoptotic activity of Jun kinase induced by tumor necrosis factor. Reuther-Madrid JY, Kashatus D, Chen S, Li X, Westwick J, Davis RJ, Earp HS, Wang CY, Baldwin Jr AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. Tumor necrosis factor (TNF) signaling through the TNF receptors involves the recruitment of key signaling factors, leading to the activation of both the transcription factor NF-kappaB and the stress-activated Jun kinase (JNK). In most cells, TNF signaling leads to a rapid and transient increase in JNK activity. However, we show that TNF treatment leads to the sustained activation of JNK in cells that are null for the p65/RelA subunit of NF-kappaB as well as in cells expressing the super-repressor form of IkappaB. In addition, the data indicate that the ability of p65/RelA to regulate gene expression is required to suppress the persistent activation of JNK. Interestingly, this suppression occurs upstream of JNK, within the signal transduction cascade leading to JNK activation, without affecting the stress-activated kinase p38. Since NF-kappaB has previously been shown to be involved in the suppression of TNF-induced apoptosis, we were interested in determining the role of deregulated JNK activity, induced by the loss of NF-kappaB, in controlling the cell death response. Through the use of different approaches for inhibition of JNK, we show that the suppression of JNK activity in cells that lack active NF-kappaB enhances the apoptotic response to TNF. These data suggest that the activity of JNK in cells blocked for NF-kappaB function provides an antiapoptotic signal and explains, at least partly, why a significant number of NF-kappaB null cells remain viable following TNF treatment. PMID: 12417721 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: 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] --------------------------------------------------------------- 35: J Immunol. 2002 Sep 1;169(5):2587-93. Inhibition of NF-kappa B activity by a membrane-transducing mutant of I kappa B alpha. Kabouridis PS, Hasan M, Newson J, Gilroy DW, Lawrence T. Bone and Joint Research Unit and Department of Experimental Pathology, Barts and London School of Medicine and Dentistry, London, United Kingdom. p.s.kabouridis@qmul.ac.uk The transcription factor NF-kappaB is regulated by the IkappaB family of proteins. The nonphosphorylatable, nondegradable superrepressor IkappaBalpha (srIkappaBalpha) mutant is a potent inhibitor of NF-kappaB activity when expressed in cells. We generated a form of srIkappaBalpha in which its N terminus is fused to the protein transduction domain of HIV TAT (TAT-srIkappaBalpha). Purified TAT-srIkappaBalpha protein rapidly and efficiently entered HeLa or Jurkat T cells. TAT-srIkappaBalpha, when exogenously added to HeLa cells, inhibited in a dose-dependent manner TNF-alpha- or IL-1beta-induced NF-kappaB activation and binding of NF-kappaB to its consensus DNA sequence. TAT-srIkappaBalpha was coimmunoprecipitated with the p65 subunit of NF-kappaB, and this interaction was resistant to stimulation with IL-1beta. Therefore, TAT-srIkappaBalpha-mediated inhibition could result from its nonreversible binding and sequestration of endogenous NF-kappaB. In contrast, exogenously added TAT-srIkappaBalpha did not inhibit IL-1beta-induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 mitogen-activated protein kinases or the phosphorylation and degradation of endogenous IkappaBalpha. These results identify a novel way for direct regulation of NF-kappaB activity in diverse cell types that may be useful for therapeutic purposes. PMID: 12193729 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Mol Biol Cell. 2002 Aug;13(8):2933-45. Cell stress and MEKK1-mediated c-Jun activation modulate NFkappaB activity and cell viability. Sanchez-Perez I, Benitah SA, Martinez-Gomariz M, Lacal JC, Perona R. Instituto de Investigaciones Biomedicas Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Spain. Chemotherapeutic agents such as cisplatin induce persistent activation of N-terminal c-Jun Kinase, which in turn mediates induction of apoptosis. By using a common MAPK Kinase, MEKK1, cisplatin also activates the survival transcription factor NFkappaB. We have found a cross-talk between c-Jun expression and NFkappaB transcriptional activation in response to cisplatin. Fibroblast derived from c-jun knock out mice are more resistant to cisplatin-induced cell death, and this survival advantage is mediated by upregulation of NFkappaB-dependent transcription and expression of MIAP3. This process can be reverted by ectopic expression of c-Jun in c-jun(-/-) fibroblasts, which decreases p65 transcriptional activity back to normal levels. Negative regulation of NFkappaB-dependent transcription by c-jun contributes to cisplatin-induced cell death, which suggests that inhibition of NFkappaB may potentiate the antineoplastic effect of conventional chemotherapeutic agents. PMID: 12181357 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Am J Physiol Gastrointest Liver Physiol. 2002 Sep;283(3):G719-26. Iron activates NF-kappaB in Kupffer cells. She H, Xiong S, Lin M, Zandi E, Giulivi C, Tsukamoto H. Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033-9141, USA. Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappaB-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappaB and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (approximately 5-50 microM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappaBalpha, a concomitant increase in nuclear p65 protein, and increased DNA binding of p50/p50 and p65/p50 without affecting activator protein-1 binding. Addition of Fe2+ to the cells resulted in an increase in electron paramagnetic resonance-detectable.OH peaking at 15 min, preceding activation of NF-kappaB but coinciding with activation of inhibitor kappaB kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappaB, and TNF-alpha promoter activity and to induce the release of TNF-alpha protein by cultured Kupffer cells in a redox status-dependent manner. We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-alpha-dependent liver injury. PMID: 12181188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: 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] --------------------------------------------------------------- 39: J Biol Chem. 2002 Mar 29;277(13):11116-25. Epub 2002 Jan 17. PTEN blocks tumor necrosis factor-induced NF-kappa B-dependent transcription by inhibiting the transactivation potential of the p65 subunit. Mayo MW, Madrid LV, Westerheide SD, Jones DR, Yuan XJ, Baldwin AS Jr, Whang YE. Department of Biochemistry, University of Virginia, Charlottesville, Virginia 22908, USA. mwm3y@virginia.edu PTEN is a lipid phosphatase responsible for down-regulating the phosphoinositide 3-kinase product phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate is involved in the activation of the anti-apoptotic effector target, Akt. Although the Akt pathway has been implicated in regulating NF-kappaB activity, it is controversial as to whether Akt activates NF-kappaB predominantly through mechanisms that regulate nuclear translocation or transactivation potential. In this report, we utilized PTEN as a natural biological inhibitor of Akt activity to study the effects on tumor necrosis factor (TNF)-induced activation of NF-kappaB. We found that the reintroduction of PTEN into prostate cells inhibited TNF-stimulated NF-kappaB transcriptional activity. PTEN failed to block TNF-induced IKK activation, IkappaBalpha degradation, p105 processing, p65 (RelA) nuclear translocation, and DNA binding of NF-kappaB. However, PTEN inhibited NF-kappaB-dependent transcription by blocking the ability of TNF to stimulate the transactivation domain of the p65 subunit. PTEN also inhibited the transactivation potential of the cyclic AMP-response element-binding protein, but this was not observed for c-Jun. The transactivation potential of p65 following TNF stimulation could be rescued from PTEN-dependent repression by re-introducing expression constructs encoding activated forms of phosphoinositide 3-kinase, Akt, or Akt and IKK. The ability of PTEN to inhibit the TNF-induced transactivation function of p65 is important, because expression of PTEN blocked TNF-stimulated NF-kappaB-dependent gene expression, thus sensitizing cells to TNF-induced apoptosis. Maintenance of the PTEN tumor suppressor protein is therefore required to modulate Akt activity and to concomitantly control the transcriptional activity of the anti-apoptotic transcription factor NF-kappaB. PMID: 11799112 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Nature. 2001 Nov 15;414(6861):313-7. Comment in: Nature. 2001 Nov 15;414(6861):265-6. Inhibition of JNK activation through NF-kappaB target genes. Tang G, Minemoto Y, Dibling B, Purcell NH, Li Z, Karin M, Lin A. Ben May Institute for Cancer Research, Committee on Cancer Biology, University of Chicago, 5841 S. Maryland Avenue, MC 6027, Chicago, Illinois 60637, USA. The proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) regulates immune responses, inflammation and programmed cell death (apoptosis). The ultimate fate of a cell exposed to TNF-alpha is determined by signal integration between its different effectors, including IkappaB kinase (IKK), c-Jun N-terminal protein kinase (JNK) and caspases. Activation of caspases is required for apoptotic cell death, whereas IKK activation inhibits apoptosis through the transcription factor NF-kappaB, whose target genes include caspase inhibitors. JNK activates the transcription factor c-Jun/AP-1, as well as other targets. However, the role of JNK activation in apoptosis induced by TNF-alpha is less clear. It is unknown whether any crosstalk occurs between IKK and JNK, and, if so, how it affects TNF-alpha-induced apoptosis. We investigated this using murine embryonic fibroblasts that are deficient in either the IKKbeta catalytic subunit of the IKK complex or the RelA/p65 subunit of NF-kappaB. Here we show that in addition to inhibiting caspases, the IKK/NF-kappaB pathway negatively modulates TNF-alpha-mediated JNK activation, partly through NF-kappaB-induced X-chromosome-linked inhibitor of apoptosis (XIAP). This negative crosstalk, which is specific to TNF-alpha signalling and does not affect JNK activation by interleukin-1 (IL-1), contributes to inhibition of apoptosis. PMID: 11713531 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Biochem Biophys Res Commun. 2001 Nov 23;289(1):25-33. Interaction of AP-1 with a cluster of NF-kappa B binding elements in the human TNF promoter region. Udalova IA, Kwiatkowski D. Wellcome Trust Centre for Human Genetics, Oxford University, Oxford OX3 7BN, United Kingdom. iudalova@molbiol.ox.ac.uk Transcriptional activation of the human TNF gene involves multiple regulatory elements whose functional properties vary between stimuli and cell types. Here we have used a COS-7 expression system to dissect the transactivating potential of NF-kappa B binding sites in the human TNF promoter region from other regulatory influences. In this model, NF-kappa B acts largely through a dense cluster of three binding sites located 600 nt upstream of the transcription start site. We show that the transcriptional activity of this complex is highly sensitive to the p65:p50 ratio that is expressed. We demonstrate that the AP-1 complex c-Jun/Fra2 is capable of binding to this region and that this inhibits the transactivating effects of NF-kappa B. These results are suggestive of a complex regulatory element that mediates fine control rather than acting as a simple on-off switch for TNF gene expression. Copyright 2001 Academic Press. PMID: 11708771 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Cancer Res. 2001 May 15;61(10):4160-8. Transformation nonresponsive cells owe their resistance to lack of p65/nuclear factor-kappaB activation. Hsu TC, Nair R, Tulsian P, Camalier CE, Hegamyer GA, Young MR, Colburn NH. Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702, USA. Clonal variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to tumor promoter-induced neoplastic transformation exhibit differential activator protein-1 (AP-1) response. Transactivation of AP-1 appears to be necessary but not sufficient to promote transformation in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibition of nuclear factor-kappaB (NF-kappaB) when transformation is suppressed, suggesting that NF-kappaB may also play a role in neoplastic transformation. We report here that transactivation of NF-kappaB is inducible by tumor promoters in P+ but not in P- JB6 cells. Inhibition of NF-kappaB using a nondegradable mutant of IkappaBalpha suppressed inducible anchorage-independent transformation of P+ JB6 cells, suggesting that NF-kappaB activation is required for tumor promotion. Induced degradation of IkappaBalpha occurred in both P+ and P- JB6 cells, indicating that failure to activate NF-kappaB in P- JB6 cells cannot be attributed to failure to degrade IkappaBalpha. Slightly higher levels of nuclear p65 were seen in P+ than in P- JB6 cells. The p65-specific DNA binding activity was also higher in P+ cells upon induction by tumor necrosis factor-alpha, suggesting that differential NF-kappaB activation may be attributable to changes in p65 activity. Transactivation of p65 protein was substantially higher in P+ than in P- JB6 cells, as determined by assay of Gal4-p65 fusion constructs. Thus activated, p65 may be a limiting factor for NF-kappaB activation and transformation responses. Stable expression of p65 in P- JB6 cells conferred not only inducible NF-kappaB and AP-1 activation but also transformation response to tumor promoters. Therefore, p65/NF-kappaB appears to be not only necessary for but also sufficient to confer tumor promotion response. Although stable expression of p65 in P- cells produced p65 increases in whole cell extracts, only the transfectants exhibiting increased nuclear p65 showed transformation response. Thus, elevation of nuclear p65 appears to be a necessary step for a transformation response. The P-/p65 transfectants showing acquired transformation response also showed elevated p65-specific transactivation response, thus recapitulating the NF-kappaB phenotypes seen in P+ cells. Expression of a transactivation-deficient mutant of Jun or dominant-negative extracellular signal-regulated kinase suppressed both AP-1 activation and p65-specific transactivation in JB6 cells, suggesting that AP-1 activity is needed for p65 transactivation and consequently for NF-kappaB activation. Thus, the transformation nonresponsive P- JB6 cells owe their resistance to lack of NF-kappaB activation and p65 transactivation that appears in turn to be attributable to insufficient AP-1 activation. PMID: 11358840 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Antioxid Redox Signal. 2000 Summer;2(2):157-75. Links between cell-surface events involving redox-active copper and gene regulation in the hemopexin heme transport system. Smith A. Division of Molecular Biology, School of Biological Sciences, University of Missouri-Kansas City, 64110-2499, USA. smithan@umkc.edu Heme is considered to play an instrumental role in the pathology of hemolysis, trauma, and reperfusion following ischemia. However, data are sparse and experimental models are required. The transport of heme by hemopexin to tissues is a specific, membrane receptor-mediated process. Hemopexin recycles after endocytosis like transferrin. Heme oxygenase-1 (HO-1), transferrin, the transferrin receptor, and ferritin are regulated by heme-hemopexin. Genes that encode proteins important for cellular defenses against oxidative stress, such as the cysteine-rich metallothioneins (MTs), are also activated by hemopexin, as are proteins that regulate cell cycle control including p21WAF1 and the tumor suppressor p53. The hemopexin system is being investigated to establish how intracellular events are affected by signal(s) from the plasma membrane due to hemopexin receptor occupancy and heme transport. A transient oxidative modification of proteins, shown by carbonyl production, takes place. Redox processes at the cell surface, which generate cuprous ions, are involved in the regulation of the MT-1 and HO-1 genes by heme-hemopexin before heme catabolism and intracellular release of iron. The "redox-sensitive" transcription factors activated by the hemopexin system include c- Jun, RelA/NFkappaB and MTF-1. The specific copper chelator bathocuproine disulfonate prevents carbonyl production, the nuclear translocation of MTF-1, and the induction of MT-1 revealing a novel, pivotal role for copper in the hemopexin system. In addition, surface redox-active copper is the first link shown for the concomitant regulation of HO-1 and MT-1 and is required for the activation of the amino-terminal c-Jun kinase (JNK) by heme-hemopexin. Publication Types: Review Review, Tutorial PMID: 11229523 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: J Immunol. 2000 Apr 15;164(8):4277-85. TNF-alpha gene expression in macrophages: regulation by NF-kappa B is independent of c-Jun or C/EBP beta. Liu H, Sidiropoulos P, Song G, Pagliari LJ, Birrer MJ, Stein B, Anrather J, Pope RM. Department of Medicine, Division of Arthritis, Veterans Administration Lakeside Medical Center, and Northwestern University Medical School, Chicago, IL 60611, USA. The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-kappa B family members interact to regulate the human TNF-alpha gene. A 120-bp TNF-alpha promoter-reporter, possessing binding sites for NF-kappa B (kappa B3), C/EBP beta (CCAAT/enhancer binding protein beta), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-kappa B p65, and c-Jun, but not C/EBP beta, suppressed (p < 0.05-0.001) LPS-induced TNF-alpha secretion in primary human macrophages. Following LPS stimulation, NF-kappa B p50/p65 heterodimers bound to the kappa B3 site and c-Jun to the -103 AP-1 site of the TNF-alpha promoter. By transient transfection, NF-kappa B p65 and p50 synergistically activated the TNF-alpha promoter. In contrast, no synergy was observed between NF-kappa B p65, with or without NF-kappa B p50, and c-Jun or C/EBP beta, even in the presence of the coactivator p300. The contribution of the upstream kappa B binding sites was also examined. Following LPS stimulation, the kappa B1 site bound both NF-kappa B p50/p65 heterodimers and p50 homodimers. The binding by NF-kappa B p50 homodimers to the kappa B1, but not to the kappa 3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent kappa B3 and AP-1 sites in the human TNF-alpha promoter contribute independently to LPS-induced activation. Although both the kappa B1 and kappa B3 sites bound transcriptionally active NF-kappa B p50/p65 heterodimers, only the kappa B1 site contributed to down-regulation by NF-kappa B p50 homodimers. PMID: 10754326 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: J Rheumatol. 2000 Dec;27(12):2769-76. Synovial fluid induced nuclear factor-kappaB DNA binding in a monocytic cell line. Lehmann T, Nguyen LQ, Handel ML. St. Vincent's Hospital Clinical School, University of New South Wales, and the Arthritis and Inflammation Research Program of the Garvan Institute of Medical Research, Sydney, Australia. OBJECTIVE: To determine the effects of synovial fluids (SF) on DNA binding activity of transcription factor nuclear factor-kappaB (NF-kappaB) in the Mono Mac 6 monocytic/macrophage cell line as a model for the interaction between SF and synovial tissue macrophages in arthritis. METHODS: Mono Mac 6 cells were incubated with SF from the knee joints of human subjects with rheumatoid arthritis (RA), undifferentiated seronegative oligoarthritis, and osteoarthritis (OA). Nuclear extracts prepared from the Mono Mac 6 cells and RA synovial tissue were analyzed by electrophoretic mobility shift analysis (EMSA) for NF-kappaB DNA binding proteins. RESULTS: Induction of NF-kappaB DNA binding by the p65(RelA)/p50 heterodimer was observed in response to incubation of Mono Mac 6 cells with SF (20% in culture medium) from 5 of 8 subjects with RA, 4 of 5 with OA, and none of 3 with undifferentiated seronegative oligoarthritis. Incubation of SF with neutralizing antibodies against tumor necrosis factor-alpha (TNF-alpha), but not antibodies against interleukin 6 (IL-6), significantly reduced the induction of p65/p50 binding activity in SF from subjects with RA and OA. Unexpectedly, a slowly migrating SF inducible NF-kappaB-binding complex was observed in EMSA of Mono Mac 6 cells after incubation with SF from 5 of 8 RA and 2 of 5 OA subjects. The induction of this complex by SF was not affected by neutralization of TNF-alpha or IL-6 in SF, and the complex was not inducible by TNF-alpha, IL-1beta, TNF-alpha/IL-1beta, IL-6, platelet derived growth factor, lipopolysaccharide, or tetradecanoyl phorbol acetate. The slowly migrating complex could not be supershifted with antibodies against NF-kappaB, Jun, or the transcriptional coactivators p300 or CBP. A NF-kappaB-binding complex with similar slow mobility was observed in nuclear extracts prepared from fresh human RA synovial tissue. CONCLUSION: Biological activity of TNF-alpha in SF from RA and OA subjects is capable of inducing p65/p50 NF-kappaB DNA binding activity in macrophages. A property of SF that is independent of TNF-alpha and other cytokines is responsible for the induction of a novel slowly migrating NF-kappaB-binding complex. Soluble mediators in SF of subjects with RA and OA can therefore modulate binding of nuclear proteins to the NF-kappaB binding site in macrophages and may play a role in inflammatory gene expression in arthritis. PMID: 11128662 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Mol Carcinog. 2000 Nov;29(3):159-69. Induced expression of dominant-negative c-jun downregulates NFkappaB and AP-1 target genes and suppresses tumor phenotype in human keratinocytes. Li JJ, Cao Y, Young MR, Colburn NH. Gene Regulation Section, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA. Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor kappaB (NFkappaB) transcription factors. The present study addresses the question of whether elevated NFkappaB in addition to elevated AP-1-dependent gene expression is necessary for maintaining the tumor cell phenotype. When a tetracycline-regulatable dominant-negative c-jun (TAM67, having a truncated transactivation domain) was expressed in tumorigenic human keratinocytes, AP-1- and NFkappaB- but not p53-dependent reporter activity was inhibited by 40-60%. Tumor phenotype, as measured by anchorage-independent growth, was inhibited by 90%. Neither AP-1/NFkappaB activation nor expression of tumor phenotype was inhibited in TAM67-harboring keratinocytes under noninducing conditions. Electrophoretic mobility shift analysis showed that induction of TAM67 expression slightly increased AP-1- but reduced NFkappaB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFkappaB p65, suggesting that inhibition of NFkappaB by TAM67 is mediated by direct protein-protein interactions, possibly producing decreased binding to DNA or inactivating p65. To analyze the putative effector genes that may be targeted by TAM67, expression of genes responsive to AP-1 or NFkappaB was measured by reverse transcriptase-polymerase chain reaction in TAM67 transfectants with or without TAM67 induction. Induction of TAM67 inhibited or reduced the expression of collagenase I, stromelysin I (AP-1 responsive), and interleukins 1 and 6 (NFkappaB responsive). These results indicate that genes controlled by NFkappaB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFkappaB activation through direct interaction with NFkappaB p65. Moreover, the findings provide proof for the principle of using inducible TAM67 as a gene therapy to suppress tumor phenotype in human carcinoma cells. PMID: 11108661 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: J Immunol. 2000 Nov 15;165(10):5788-97. Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe Universitat, Frankfurt am Main, Germany. The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1beta-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2, 3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1beta-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5'-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1beta-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-kappaB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1beta. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1beta-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-kappaB. PMID: 11067938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Biochem Biophys Res Commun. 2000 Jul 21;274(1):4-10. Suppression of IkappaBalpha expression is necessary for c-Jun N-terminal kinase-mediated enhancement of Fas cytotoxicity. Chang NS, Schultz L, Heath J. Laboratory of Molecular Immunology, Guthrie Research Institute, Guthrie Medical Center, 1 Guthrie Square, Sayre, Pennsylvania 18840, USA. nschang@inet.guthrie.org The role of c-Jun N-terminal kinase (JNK) in the regulation of Fas-mediated cell death was investigated. Murine L929 fibroblasts were pretreated with anisomycin for 1 h to activate JNK, followed by exposure to anti-Fas antibodies/actinomycin D (ActD) for 16-24 h. Compared to untreated controls, the induction of JNK activation failed to raise cellular sensitivity to anti-Fas/ActD killing. Notably, a significant increase in anti-Fas/ActD killing as induced by JNK preactivation was observed in L929 cells which were engineered to suppress IkappaBalpha protein expression by antisense mRNA. Restoration of the IkappaBalpha protein level in these cells by ectopic expression of a cDNA construct abolished the JNK-increased anti-Fas/ActD killing. Despite the suppression of IkappaBalpha, no constitutive p65 (RelA) NF-kappaB nuclear translocation was observed in the IkappaBalpha-antisense cells. Also, inhibition of NF-kappaB by curcumin failed to inhibit the JNK-increased Fas cytotoxicity, suggesting that NF-kappaB is not involved in the observed effect. Most interestingly, culturing of L929 cells on extracellular protein matrices resulted in partial suppression of IkappaBalpha expression and constitutive JNK and p42/44 MAPK activation. Upon stimulation with anisomycin, these matrix protein-stimulated cells further exhibited reduced IkappaBalpha expression and p42/44 MAPK activation, as well as became sensitized to JNK-increased anti-Fas/ActD killing. Again, ectopic expression of IkappaBalpha in these cells abolished the enhanced anti-Fas/ActD killing effect. Together, these results indicate that suppression of IkappaBalpha expression is essential for JNK-mediated enhancement of Fas cytotoxicity. Copyright 2000 Academic Press. PMID: 10903887 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: J Immunol. 2000 Jun 15;164(12):6509-19. Resveratrol suppresses TNF-induced activation of nuclear transcription factors NF-kappa B, activator protein-1, and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. Manna SK, Mukhopadhyay A, Aggarwal BB. Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin found in grapes, fruits, and root extracts of the weed Polygonum cuspidatum, exhibits anti-inflammatory, cell growth-modulatory, and anticarcinogenic effects. How this chemical produces these effects is not known, but it may work by suppressing NF-kappaB, a nuclear transcription factor that regulates the expression of various genes involved in inflammation, cytoprotection, and carcinogenesis. In this study, we investigated the effect of resveratrol on NF-kappaB activation induced by various inflammatory agents. Resveratrol blocked TNF-induced activation of NF-kappaB in a dose- and time-dependent manner. Resveratrol also suppressed TNF-induced phosphorylation and nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB-dependent reporter gene transcription. Suppression of TNF-induced NF-kappaB activation by resveratrol was not restricted to myeloid cells (U-937); it was also observed in lymphoid (Jurkat) and epithelial (HeLa and H4) cells. Resveratrol also blocked NF-kappaB activation induced by PMA, LPS, H2O2, okadaic acid, and ceramide. The suppression of NF-kappaB coincided with suppression of AP-1. Resveratrol also inhibited the TNF-induced activation of mitogen-activated protein kinase kinase and c-Jun N-terminal kinase and abrogated TNF-induced cytotoxicity and caspase activation. Both reactive oxygen intermediate generation and lipid peroxidation induced by TNF were suppressed by resveratrol. Resveratrol's anticarcinogenic, anti-inflammatory, and growth-modulatory effects may thus be partially ascribed to the inhibition of activation of NF-kappaB and AP-1 and the associated kinases. PMID: 10843709 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: J Immunol. 2000 Jun 1;164(11):5815-25. Vesnarinone suppresses TNF-induced activation of NF-kappa B, c-Jun kinase, and apoptosis. Manna SK, Aggarwal BB. Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. Vesnarinone, a synthetic quinolinone derivative used in the treatment of cardiac failure, exhibits immunomodulatory, anti-inflammatory, and cell growth regulatory properties. The mechanisms underlying these properties are not understood, but due to the critical role of nuclear transcription factor NF-kappa B in these responses, we hypothesized that vesnarinone must modulate NF-kappa B activation. We investigated the effect of vesnarinone on NF-kappa B activation induced by inflammatory agents. Vesnarinone blocked TNF-induced activation of NF-kappa B in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of I kappa B alpha, an inhibitor of NF-kappa B. The effects of vesnarinone were not cell type specific, as it blocked TNF-induced NF-kappa B activation in a variety of cells. NF-kappa B-dependent reporter gene transcription activated by TNF was also suppressed by vesnarinone. The TNF-induced NF-kappa B activation cascade involving TNF receptor 1-TNF receptor associated death domain-TNF receptor associated factor 2 NF-kappa B-inducing kinase-IKK was interrupted at the TNF receptor associated factor 2 and NF-kappa B-inducing kinase sites by vesnarinone, thus suppressing NF-kappa B reporter gene expression. Vesnarinone also blocked NF-kappa B activation induced by several other inflammatory agents, inhibited the TNF-induced activation of transcription factor AP-1, and suppressed the TNF-induced activation of c-Jun N-terminal kinase and mitogen-activated protein kinase kinase. TNF-induced cytotoxicity, caspase activation, and lipid peroxidation were also abolished by vesnarinone. Overall, our results indicate that vesnarinone inhibits activation of NF-kappa B and AP-1 and their associated kinases. This may provide a molecular basis for vesnarinone's ability to suppress inflammation, immunomodulation, and growth regulation. PMID: 10820260 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: 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] --------------------------------------------------------------- 52: J Neurochem. 2000 Feb;74(2):527-39. Functional interplay between nuclear factor-kappaB and c-Jun integrated by coactivator p300 determines the survival of nerve growth factor-dependent PC12 cells. Maggirwar SB, Ramirez S, Tong N, Gelbard HA, Dewhurst S. Department of Microbiology and Immunology, University of Rochester Medical Center, New York 14642, USA. sanjay_maggirwar@urmc.rochester.edu Nerve growth factor (NGF) activates the transcription factors nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) in sympathetic neurons. Whereas NGF-inducible NF-kappaB is required for the survival of neurons, c-Jun has the ability to promote neuronal death. In this report, we have examined the effect of NGF withdrawal on c-Jun and NF-kappaB transcription factors in PC12 cells differentiated to a neuronal phenotype. We show that the withdrawal of NGF from these cultures results in de novo synthesis of c-Jun, increase in AP-1 activity, and down-regulation of NF-kappaB activity. To investigate how the signal transduction pathways activating c-Jun and NF-kappaB are differentially regulated by NGF, we performed transcriptional analyses. Expression of ReIA (NF-kappaB) suppressed the c-Jun-dependent transcription of c-jun, and this effect was reversed by overexpression of the coactivator p300. RelA's effects on c-Jun transcription were mediated by competitive binding of the carboxy-terminal region of RelA to the CH1 domain of p300, which also binds to c-Jun; deletion of this region abrogated the ability of RelA to inhibit c-Jun activity. Furthermore, the inhibition of endogenous NF-kappaB in NGF-maintained neuronal PC12 cells led to the induction of c-Jun synthesis and a marked increase in cell death. Together, these studies demonstrate a functional interaction between NF-kappaB and c-Jun and suggest a novel mechanism of NF-kappaB-mediated neuroprotection. PMID: 10646503 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Immunol. 1999 Dec 15;163(12):6800-9. Silymarin suppresses TNF-induced activation of NF-kappa B, c-Jun N-terminal kinase, and apoptosis. Manna SK, Mukhopadhyay A, Van NT, Aggarwal BB. Department of Molecular Oncology, Cytokine Research Laboratory, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-inflammatory, cytoprotective, and anticarcinogenic effects. How silymarin produces these effects is not understood, but it may involve suppression of NF-kappa B, a nuclear transcription factor, which regulates the expression of various genes involved in inflammation, cytoprotection, and carcinogenesis. In this report, we investigated the effect of silymarin on NF-kappa B activation induced by various inflammatory agents. Silymarin blocked TNF-induced activation of NF-kappa B in a dose- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of Iota kappa B alpha, an inhibitor of NF-kappa B. Silymarin blocked the translocation of p65 to the nucleus without affecting its ability to bind to the DNA. NF-kappa B-dependent reporter gene transcription was also suppressed by silymarin. Silymarin also blocked NF-kappa B activation induced by phorbol ester, LPS, okadaic acid, and ceramide, whereas H2O2-induced NF-kappa B activation was not significantly affected. The effects of silymarin on NF-kappa B activation were specific, as AP-1 activation was unaffected. Silymarin also inhibited the TNF-induced activation of mitogen-activated protein kinase kinase and c-Jun N-terminal kinase and abrogated TNF-induced cytotoxicity and caspase activation. Silymarin suppressed the TNF-induced production of reactive oxygen intermediates and lipid peroxidation. Overall, the inhibition of activation of NF-kappa B and the kinases may provide in part the molecular basis for the anticarcinogenic and anti-inflammatory effects of silymarin, and its effects on caspases may explain its role in cytoprotection. PMID: 10586080 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: 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] --------------------------------------------------------------- 55: 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] --------------------------------------------------------------- 56: Cell Signal. 1999 May;11(5):371-83. Dual activity of pyrrolidine dithiocarbamate on kappaB-dependent gene expression in U937 cells: II. Regulation by tumour necrosis factor-alpha. Watanabe K, Kazakova I, Furniss M, Miller SC. Signal Transduction Laboratory, Pharmaceutical Discovery Division, SRI International, Menlo Park, CA 94025, USA. In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-kappaB (NF-kappaB) signalling pathway induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-alpha (TNF-alpha)-induced NF-kappaB DNA binding activity but potentiated the effect of TNF-alpha on kappaB-dependent gene expression. The stimulatory effect of PDTC with TNF-alpha was not observed with an HIV-1 LTR reporter construct containing two mutated kappaB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two kappaB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-alpha, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on kappaB-dependent gene expression. Evidence that PDTC induced AP-1 DNA binding and AP-1 reporter gene activity, raised the hypothesis that the effect of PDTC was mediated by an interaction between the AP-1 pathway and p65(RelA). Co-transfection with expression vectors for p65(RelA) and the AP-1 subunits c-Fos and c-Jun resulted in a decrease in the stimulatory effect of PDTC on HIV-1 LTR activity. Co-transfection of p65(RelA) with Tam67, a dominant negative mutant of c-Jun defective in transactivation, stimulated the effect of PDTC on HIV-1 LTR activity. Evidence that the stimulatory effect of Tam67 with PDTC was reduced with c-Jun is consistent with the hypothesis. PMID: 10376811 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: J Immunol. 1999 May 1;162(9):5337-44. NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells. Awane M, Andres PG, Li DJ, Reinecker HC. Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA 02114, USA. IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells. PMID: 10228009 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: J Immunol. 1999 Apr 1;162(7):4226-34. Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts. Kouba DJ, Chung KY, Nishiyama T, Vindevoghel L, Kon A, Klement JF, Uitto J, Mauviel A. Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA. Among its plethora of activities as an inflammatory mediator, TNF-alpha has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-alpha response element of the COL1A2 promoter. The TNF-alpha response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was eliminated by a 2-bp substitution mutation in the NF-kappa B1 binding half site of the NF-kappa B cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-alpha stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-kappa B (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-alpha activates and initiates the nuclear translocation of NF-kappa B that binds a divergent NF-kappa B element and plays a critical role in the observed inhibition of alpha 2(I) collagen gene transcription. PMID: 10201951 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: Biochem Pharmacol. 1999 Apr 1;57(7):763-74. Suppression of tumor necrosis factor-activated nuclear transcription factor-kappaB, activator protein-1, c-Jun N-terminal kinase, and apoptosis by beta-lapachone. Manna SK, Gad YP, Mukhopadhyay A, Aggarwal BB. Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. Beta-lapachone, the product of a tree from South America, is known to exhibit various pharmacologic properties, the mechanisms of which are poorly understood. In the present report, we examined the effect of beta-lapachone on the tumor necrosis factor (TNF)-induced activation of the nuclear transcription factors NF-kappaB and activator protein-1 (AP-1) in human myeloid U937 cells. TNF-induced NF-kappaB activation, p65 translocation, IkappaBalpha degradation, and NF-kappaB-dependent reporter gene expression were inhibited in cells pretreated with beta-lapachone. Direct treatment of the p50-p65 heterodimer of NF-kappaB with beta-lapachone had no effect on its ability to bind to the DNA. Besides myeloid cells, beta-lapachone was also inhibitory in T-cells and epithelial cells. Beta-lapachone also suppressed the activation of NF-kappaB by lipopolysaccharide, okadaic acid, and ceramide but had no significant effect on activation by H2O2 or phorbol myristate acetate, indicating that its action is selective. Beta-lapachone also abolished TNF-induced activation of AP-1, c-Jun N-terminal kinase, and mitogen-activated protein kinase kinase (MAPKK or MEK). TNF-induced cytotoxicity and activation of caspase-3 were also abolished by beta-lapachone. Because reducing agents (dithiothreitol and N-acetylcysteine) reversed the effect of beta-lapachone, it suggests the role of a critical sulfhydryl group. Overall, our results identify NF-kappaB, AP-1, and apoptosis as novel targets for beta-lapachone, and this may explain some of its pharmacologic effects. PMID: 10075082 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Autoimmunity. 1998;28(4):197-208. AP-1 and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis. Han Z, Boyle DL, Manning AM, Firestein GS. Division of Rheumatology, UCSD School of Medicine, La Jolla, CA 92093-0656, USA. OBJECTIVE: To determine the expression and regulation of nuclear transcription factors AP-1 and NF-kappaB in rheumatoid arthritis and in collagen-induced arthritis in mice. METHODS: AP-1 and NF-kappaB expression and function were determined in RA, OA and normal synovial tissue by electrophoretic mobility shift assay (EMSA) and immunohistochemistry. The kinetics of transcription factor expression were then examined in collagen-induced arthritis (CIA) in mice. EMSAs were performed with the nuclear extracts obtained from paws of CIA mice from 10 to 45d after immunization to determine AP-1 and NF-kappaB binding activity. The expression of collagenase-3 (MMP13) and stromelysin (MMP3) mRNA was examined by northern blot analysis. RESULTS: Immunohistochemistry showed that NF-kappaB expression was increased in both RA and OA synovial intimal lining. AP-1 components Jun and Fos were also present in the intimal lining and was significantly greater in RA than OA. The DNA binding activities of both AP-1 and NF-kappaB were significantly higher RA patients compared with OA. In CIA, AP-1 and NF-kappaB expression increased by day 20, which was 1-2 weeks before onset of clinical arthritis. However, collagenase and stromelysin gene expression did not increase until day 35. CONCLUSION: The DNA binding activity of AP-1 and NF-kappaB are markedly increased in both CIA and RA. In CIA, activation of AP-1 and NF-kappaB precede both clinical arthritis and metalloproteinase gene expression. NF-kappaB expression correlated better than AP-1 with metalloproteinase expression. PMID: 9892501 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Oncogene. 1998 Dec 3;17(22):2889-99. Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53. Herrmann JL, Briones F Jr, Brisbay S, Logothetis CJ, McDonnell TJ. The Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston 77030, USA. The ATP/ubiquitin-dependent 26S proteasome is a central regulator of cell cycle progression and stress responses. While investigating the application of peptide aldehyde proteasome inhibitors to block signal-induced IkappaBalpha degradation in human LNCaP prostate carcinoma cells, we observed that persistent inhibition of proteasomal activity signals a potent cell death program. Biochemically, this program included substantial upregulation of PAR-4 (prostate apoptosis response-4), a putative pro-apoptotic effector protein and stabilization of c-jun protein, a potent pro-death effector in certain cells. We also observed modest downregulation of bcl-XL, a pro-survival effector protein. However, in contrast to some recent reports stable, high level, expression of functional bcl-2 protein in prostate carcinoma cells failed to signal protection against cell death induction by proteasome inhibitors. Also in disagreement to a recent report, no evidence was found for activation of the JNK stress kinase pathway. A role for p53, a protein regulated by the proteasome pathway, was ruled out, since comparable cell death induction by proteasome inhibitors occurred in PC-3 cells that do not express functional p53 protein. These data signify that the ubiquitin/proteasome pathway represents a potential therapeutic target for prostate cancers irrespective of bcl-2 expression or p53 mutations. PMID: 9879995 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: 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] --------------------------------------------------------------- 63: J Neurosci Res. 1998 Nov 15;54(4):465-74. NGF induces apoptosis in a human neuroblastoma cell line expressing the neurotrophin receptor p75NTR. Kuner P, Hertel C. F. Hoffmann-LaRoche Ltd., Pharma Division, Preclinical Research, Basel, Switzerland. Nerve growth factor (NGF) has been demonstrated to support survival and differentiation of neuronal cells. Recently, a role of NGF in neuronal apoptosis has been suggested. NGF binds to tropomyosin receptor kinase A (TrkA) and to 75-kDa NGF receptor (p75NTR). TrkA is responsible for differentiation and survival, but p75NTR, a member of the death receptor family, seems to mediate the apoptotic effect of NGF. Here we demonstrate that NGF-but not neurotrophin-3 (NT-3) or brain-derived neurotrophic factor (BDNF)-induced apoptosis in p75NTR-expressing human neuroblastoma SK-N-MC cells. BDNF prevented NGF-induced apoptosis. NGF-induced apoptosis was accompanied by the release of NFkappaB p65 and the activation of stress-activated protein kinase/c-jun amino terminal kinase. Because p75NTR and NGF are upregulated in Alzheimer's disease, NGF/p75NTR might be involved in neuronal cell death related to the disease. PMID: 9822157 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: J Immunol. 1998 Sep 15;161(6):2863-72. IL-13 suppresses TNF-induced activation of nuclear factor-kappa B, activation protein-1, and apoptosis. Manna SK, Aggarwal BB. Department of Molecular Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. IL-13 is known to suppress the production of inflammatory cytokines such as TNF. Whether IL-13 also modulates the biologic effects of TNF is not known. In the present report we examined the effect of IL-13 on TNF-induced activation of nuclear transcription factors NF-kappa B and activation protein-1 (AP-1) and apoptosis. Pretreatment of cells with IL-13 blocked TNF-induced NF-kappa B activation, nuclear translocation of p65 subunit, and degradation of I kappa B alpha. IL-13 also inhibited NF-kappa B activation by LPS, okadaic acid, H2O2, and ceramide. TNF-induced NF-kappa B-dependent gene transcription was also blocked by IL-13. TNF-induced activation of another nuclear transcription factor, AP-1, was suppressed by IL-13. The activation of N-terminal c-Jun kinase and mitogen-activated protein kinase kinase, implicated in the regulation of AP-1 and NF-kappa B, was also down-regulated by IL-13. TNF-mediated cytotoxicity and activation of caspase-3 were abolished by IL-13. The inhibitory effects of IL-13 on TNF were sensitive to H-7, neomycin, and wortmannin, suggesting that the pathway consisting of protein kinase C, phosphatidylinositol 3-kinase, and phospholipase C must be involved in IL-13 signaling. Thus, overall, these results demonstrate that IL-13 is a potent inhibitor of TNF-mediated activation of NF-kappa B, AP-1, and apoptosis, which may contribute to its previously described immunosuppressive and anti-inflammatory effects. PMID: 9743347 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Mol Cell. 1998 Jan;1(2):277-87. Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Merika M, Williams AJ, Chen G, Collins T, Thanos D. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA. Transcriptional activation of the IFN beta gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappa B, IRF1, ATF2/c-Jun, and the architectural protein HMG I(Y). The level of transcription generated by all of these activators is greater than the sum of the levels generated by individual factors, a phenomenon designated transcriptional synergy. We demonstrate that this synergy, in the context of the enhanceosome, requires a new protein-protein interaction domain in the p65 subunit of NF-kappa B. Transcriptional synergy requires recruitment of the CBP/p300 coactivator to the enhanceosome, via a new activating surface assembled from the novel p65 domain and the activation domains of all of the activators. Deletion, substitution, or rearrangement of any one of the activation domains in the context of the enhanceosome decreases both recruitment of CBP and transcriptional synergy. PMID: 9659924 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Mol Cell. 1997 Dec;1(1):119-29. The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome. Kim TK, Maniatis T. Harvard University, Department of Molecular and Cellular Biology, Cambridge, Massachusetts 02138, USA. A functional interferon-beta gene enhanceosome was assembled in vitro using the purified recombinant transcriptional activator proteins ATF2/c-JUN, IRF1, and p50/p65 of NF-kappa B. Maximal levels of transcriptional synergy between these activators required the specific interactions with the architectural protein HMG I(Y) and the correct helical phasing of the binding sites of these proteins on the DNA helix. Analyses of the in vitro assembled enhanceosome revealed that the transcriptional synergy is due, at least in part, to the cooperative assembly and stability of the complex. Reconstitution experiments showed that the formation of a stable enhanceosome-dependent preinitiation complex require cooperative interactions between the enhanceosome; the general transcription factors TFID, TFIIA, and TFIIB; and the cofactor USA. These studies provide a direct biochemical demonstration of the importance of the structure and function of natural multicomponent transcriptional enhancer complexes in gene regulation. PMID: 9659909 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: EMBO J. 1998 May 15;17(10):2846-54. Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. Gunes C, Heuchel R, Georgiev O, Muller KH, Lichtlen P, Bluthmann H, Marino S, Aguzzi A, Schaffner W. Institut fur Molekularbiologie, Abteilung II der Universitat Zurich, Zurich, Switzerland. We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA). PMID: 9582278 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: 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] --------------------------------------------------------------- 69: 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] --------------------------------------------------------------- 70: Gene Expr. 1997;6(6):361-70. Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kappaB family members. Ma S, Rao L, Freedberg IM, Blumenberg M. The Ronald O. Perelman Department of Dermatology, New York University Medical Center, NY 10016, USA. The expression of keratins K5 and K14 is restricted to the basal layers of the healthy epidermis, whereas the expression of K6 and K17 is induced in response to proliferative and inflammatory signals, respectively. The control of keratin expression occurs primarily at the transcriptional level. We studied the effects of transcription factors of the AP-1 and NF-kappaB families on the expression of those four keratin genes. We chose AP-1 and NF-kappaB proteins because they are activated by many extracellular signals, including those in hyperproliferative and inflammatory processes. DNA constructs expressing the transcription factors were, in various combinations, cotransfected with constructs containing keratin gene promoters and the CAT reporter gene into HeLa cells or keratinocytes. We found that the K5 and K14 promoters, which are coexpressed in vivo, are regulated in parallel by the cotransfected genes. Both were activated by the c-Fos and c-Jun components of AP-1, but not by Fra1. On the other hand, the NF-kappaB proteins, especially p65, suppressed these two promoters. The K17 promoter was specifically activated by c-Jun, whereas the other transcription factors tested had no significant effect. In contrast, the K6 promoter was very strongly activated by all AP-1 proteins, especially by the c-Fos + c-Jun and Fra1 + c-Jun combinations. It was also strongly activated by the p65 NF-kappaB protein. AP-1 and NF-kappaB acted synergistically in activating the K6 promoter, although the AP-1 and the NF-kappaB responsive sites could be separated physically. These results suggest that the interplay of AP-1 and NF-kappaB proteins regulates epidermal gene expression and that the activation of these transcription factors by extracellular signaling molecules brings about the differential expression of keratin genes in epidermal differentiation, cutaneous diseases, and wound healing. PMID: 9495317 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: 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] --------------------------------------------------------------- 72: 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] --------------------------------------------------------------- 73: Oncogene. 1997 Jun 12;14(23):2845-55. ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter. Thomas RS, Tymms MJ, McKinlay LH, Shannon MF, Seth A, Kola I. Molecular Genetics and Development Group, Institute of Reproduction and Development, Monash University, Melbourne, Australia. Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM-CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkappaB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GM-CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM-CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM-CSF reporter construct in unstimulated Jurkat cells, providing that either NFkappaB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFkappaB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkappaB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkappaB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells. PMID: 9190901 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: J Biol Chem. 1997 Mar 28;272(13):8558-66. Transcription factor activation during signal-induced apoptosis of immature CD4(+)CD8(+) thymocytes. A protective role of c-Fos. Ivanov VN, Nikolic-Zugic J. Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. Many signals that cause apoptotic cell death operate by inducing transcription and translation of other (presumably death effector) mediators, and it is well established that stimulus-induced apoptosis can often be blocked by inhibiting transcription and translation. Transcriptional regulation of apoptosis, however, is incompletely understood. To gain insight into nuclear events associated with signal-induced apoptosis during T cell development, we studied signal-induced apoptosis of ex vivo isolated immature CD8(+)4(+) double-positive (DP) thymocytes. Stimuli utilizing the T cell receptor (TCR) signaling pathway or its parts (an alphaCD3/TCR monoclonal antibody, a Ca2+ ionophore, or a protein kinase C-activating phorbol ester) or a stimulus that antagonizes TCR signaling and apoptosis in T cell hybridoma (forskolin, a cyclic AMP-signaling activator) resulted in massive apoptosis of DP thymocytes. At the same time, these stimuli induced qualitatively similar but quantitatively unique patterns of inducible transcription factors (TFs) NF-kappaB/RelA-p50, AP-1 (Fos-Jun), and NUR-77. We focused our attention on the role of AP-1 (Fos-Jun) complex, which was strongly induced by all of the above stimuli and thus was a candidate for a proapoptotic TF. However, we found that AP-1/c-Fos induction was vital in prolonging DP thymocyte life, as judged by increased spontaneous and induced death of DP cells in Fos-/- mice. In direct support of this hypothesis, experiments with antisense oligonucleotides demonstrated that c-Fos plays an essential role in protecting normal DP thymocytes from Ca2+- and cAMP-induced apoptosis but not from TCR-mediated death. Together, these results demonstrate a physiological role for c-Fos in maintaining longevity of DP thymocytes. PMID: 9079686 [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: Mol Cell Biol. 1996 Jan;16(1):359-68. The interferon-inducible p202 protein as a modulator of transcription: inhibition of NF-kappa B, c-Fos, and c-Jun activities. Min W, Ghosh S, Lengyel P. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA. The antimicrobial, immunomodulatory, and cell growth-regulatory activities of the interferons are mediated by interferon-inducible proteins. One of these is p202, a nuclear protein that is encoded by the Ifi 202 gene from the interferon-activatable gene 200 cluster. Overexpression of p202 in transfected cells slows down cell proliferation. As shown earlier, p202 binds to the hypophosphorylated form of the retinoblastoma susceptibility protein. Here we report that p202 inhibits the activities of the NF-kappa B and the AP-1 enhancers both in transiently transfected cells and in transfected stable cell lines overexpressing p202. Furthermore, p202 binds the NF-kappa B p50 and p65 and the AP-1 c-Fos and c-Jun transcription factors in vitro and in vivo. NF-kappa B, c-Fos, and c-Jun participate in the transcription of various cellular and viral genes, and thus p202 can modulate the expression of these genes in response to interferons. PMID: 8524315 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Arthritis Rheum. 1995 Dec;38(12):1762-70. Nuclear factor-kappa B in rheumatoid synovium. Localization of p50 and p65. Handel ML, McMorrow LB, Gravallese EM. Harvard School of Public Health, Boston, Massachusetts, USA. OBJECTIVE: To identify the cells that express transcription factor NF-kappa B subunits p50 and p65 in synovial tissue from patients with rheumatoid arthritis (RA) and to correlate the distribution of p50 and p65 with CD14 (macrophage lipopolysaccharide receptor) and members of the AP-1 transcription factor family, Jun and Fos. METHODS: Immunohistochemistry was used to identify p50, p65, Jun and Fos in sections of synovial tissue from 13 patients with RA and 4 "normal" control subjects. Double staining for CD14 and each of the transcription factor subunits was performed. RESULTS: Subunits p50 and p65 were present in the nuclei of synovial cells in all 13 RA patients, with expression varying from rare cells to more than half of all cells. In most cases, nuclear p50 and p65 were present in approximately one-third of synovial lining cells and in a variable proportion of cells scattered throughout the sublining region, including the endothelium. The distributions of p50 and p65 were similar. Jun and Fos were present in the nuclei of a large proportion of synovial lining cells with significantly less expression elsewhere. In each case the Jun/Fos distribution was clearly different from the p50/p65 distribution, although there was significant overlap in many cases. Cells expressing CD14 were mostly Jun/Fos negative and were predominantly p50/p65 positive. There was negligible staining for p50 or p65 in the 4 normal control synovium samples. CONCLUSION: In most RA patients, the p50 and p65 subunits of NF-kappa B were present in the majority of CD14-positive cells within the lining and sublining regions and in a proportion of other cells throughout the synovium, including endothelial cells. NF-kappa B is likely to play an important role in the expression of macrophage-derived cytokines in rheumatoid synovium. Different but overlapping distributions of nuclear p50 and p65 versus Jun and Fos indicate separate or divergent mechanisms for the activation of NF-kappa B and the expression of AP-1 proteins in rheumatoid synovium. PMID: 8849348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: 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] --------------------------------------------------------------- 79: J Immunol. 1994 Sep 15;153(6):2394-406. Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E. Ivanov V, Fleming TJ, Malek TR. Department of Microbiology and Immunology, University of Miami School of Medicine, FL 33101. Cross-linking of glycosylphosphatidylinositol-anchored proteins, including mouse Ly-6A/E, leads to IL-2 secretion and T cell activation, whereas engagement of Ly-6A/E uniquely inhibits IL-2 production induced via TCR. However, little is known concerning the molecular mechanism by which glycosylphosphatidylinositol-anchored proteins regulate IL-2 expression. In this study, we have examined the ability of an anti-Ly-6A/E mAb to regulate transcription factors controlling IL-2 expression. Stimulation of EL4J(Ly-6E).A4 cells with anti-CD3 epsilon or anti-Ly6A/E mAbs induced nuclear factor (NF)-kappa B p65-p50 (RelA/p50) and AP-1 (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-Y levels were stable. Cyclic AMP response element binding protein and T cell-specific factor-1 (alpha) activities were selectively enhanced by anti-CD3 epsilon, but not by anti-Ly6A/E, which suggests that signaling via the TCR and Ly-6 were not identical. Costimulation of these cells with both mAbs produced substantially reduced levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 whereas cyclic AMP response element binding protein and T cell-specific factor-1(alpha) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the IL-2 enhancer in vivo and the contribution of individual transcription factors for this induction were assessed with use of reporter chloramphenicol acetyltransferase constructs containing the IL-2 enhancer or oligomerized binding sites for transcription factors. These experiments also demonstrated a key role for NF-kappa B and AP-1 in the transcriptional regulation of the IL-2 gene by TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybridoma and a mutated variant, were revealed a crucial role for the zeta-chain in Ly6A/E-mediated activation of NF-kappa B. PMID: 7915738 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------