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• W1549679997 abstract "Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-κB plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-κB-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple κB-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a κB-dependent reporter gene construct, without affecting the levels of NF-κB binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 “one-hybrid” system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 κB subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-κB, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery. Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-κB plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-κB-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple κB-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a κB-dependent reporter gene construct, without affecting the levels of NF-κB binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 “one-hybrid” system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 κB subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-κB, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery. Interleukin (IL) 1The abbreviations used are: IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; CBP, cAMP responsive element binding protein binding protein; Tricine, N-tris(hydroxymethyl)methylglycine. 1The abbreviations used are: IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; CBP, cAMP responsive element binding protein binding protein; Tricine, N-tris(hydroxymethyl)methylglycine. 6 contributes to a multitude of physiological and pathophysiological processes. Among its many functions, IL-6 plays an active role in immunological responses, bone metabolism, reproduction, inflammation, neoplasia, and aging. The cellular and molecular biology of IL-6 has been explored by a variety of approaches (1Hirano T. Kishimoto T. Res. Immunol. 1992; 143: 689-788Google Scholar, 2Keller E.T. Wanagat J. Ershler W.B. Frontiers Biosci. 1996; 1: d340-d357Crossref PubMed Scopus (124) Google Scholar). The regulation of expression of the IL-6 gene is adapted to the key function of this cytokine, namely a systemic alarm signal that recruits diverse host defense mechanisms that serve to limit tissue injury. Inflammation-associated cytokines such as tumor necrosis factor (TNF), IL-1, and platelet-derived growth factor, bacterial products such as endotoxin, and acute viral infections, all enhance IL-6 gene expression. The characterization of the IL-6 promoter revealed a complex control region that can be triggered by multiple activation pathways (3Dendorfer U. Oettgen P. Libermann T.A. Mol. Cell. Biol. 1994; 14: 4443-4454Crossref PubMed Scopus (298) Google Scholar, 4Haegeman G. Fiers W. Packer L. Wirtz K. Signalling Mechanisms: From Transcription Factors to Oxidative Stress. Springer Verlag, Berlin1995: 375-382Google Scholar).In the case of TNF (and of IL-1), the main transcriptional activator for IL-6 gene induction is the nuclear factor (NF)-κB (5Libermann T.A. Baltimore D. Mol. Cell. Biol. 1990; 10: 2327-2334Crossref PubMed Google Scholar, 6Shimizu H. Mitomo K. Watanabe T. Okamoto S. Yamamoto K. Mol. Cell. Biol. 1990; 10: 561-568Crossref PubMed Scopus (316) Google Scholar, 7Zhang Y. Lin J.-X. Vilček J. Mol. Cell. Biol. 1990; 10: 3818-3823Crossref PubMed Scopus (233) Google Scholar), which is typically a dimer between p50 and the transactivating subunit p65 (RelA). In unstimulated cells, NF-κB resides in the cytoplasm. Here, the DNA-binding dimer is bound to the inhibitory molecule IκB, from which it is released upon cell stimulation. NF-κB then migrates into the nucleus, where it effects expression of its numerous target genes (8Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4582) Google Scholar).We have shown previously that in the mouse fibrosarcoma cell line L929sA, the quantities of activated NF-κB do not correlate with the variations of IL-6 mRNA levels in the cell. We therefore concluded that other cooperative factors or regulatory mechanisms are necessary for modulating the levels of NF-κB-dependent IL-6 mRNA production (9Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar).Studies over the last few years have shown that different mitogen-activated kinase (MAPK) cascade pathways contribute to the transmission of extracellular signals that can finally result in direct or indirect phosphorylation of various transcription factors and alterations in gene expression (10Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1160) Google Scholar, 11Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). More particularly, we reported that abrogation of the p38 MAPK pathway represses TNF-mediated IL-6 gene expression, but not NF-κB activation and DNA binding (12Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar).In the present article, we report on the essential role of NF-κB to trigger IL-6 gene activation in response to TNF in L929sA cells. Furthermore, we show that, apart from TNF-induced cytoplasmic NF-κB activation and nuclear DNA binding, the TNF-activated p38 and ERK MAPK pathways contribute to transcriptional activation by modulating the transactivation capacity of the NF-κB p65 subunit.DISCUSSIONIn this paper, we have focused on the transcriptional activation by the factor NF-κB in response to TNF. By the use of various deleted and point-mutated versions of the IL-6 promoter, we have documented the key role of the κB motif for induction by TNF. A number of previous reports have already described the necessity of cooperation and association of NF-κB with other DNA-bound transcription factors for optimal gene activation (33Kaszubska W. Hooft van Huijsduijnen R. Ghersa P. DeRaemy-Schenk A.-M. Chen B.P.C. Hai T. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1993; 13: 7180-7190Crossref PubMed Google Scholar, 34Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (873) Google Scholar, 35Stein B. Baldwin Jr., A.S. Ballard D.W. Greene W.C. Angel P. Herrlich P. EMBO J. 1993; 12: 3879-3891Crossref PubMed Scopus (567) Google Scholar, 36Ray A. Hannink M. Ray B.K. J. Biol. Chem. 1995; 270: 7365-7374Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The deletion analysis of the IL-6 promoter shows that such factors have indeed a co-activating and integrating function for full stimulation of the IL-6 promoter. However, using “loss-of-function” mutants of the IL-6 promoter, the crucial role of NF-κB is obvious, without the primary need for other associating DNA-bound factors. Using the “gain-of-function” approach by inserting multiple κB sites in front of an unresponsive promoter, the TNF response could be restored.Activation of NF-κB and its binding to DNA is, however, not sufficient for IL-6 gene activation by TNF; the requirement of additional activating mechanisms has already been described previously (4Haegeman G. Fiers W. Packer L. Wirtz K. Signalling Mechanisms: From Transcription Factors to Oxidative Stress. Springer Verlag, Berlin1995: 375-382Google Scholar, 9Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar). More particularly, recently, we have established the importance of the p38 MAPK pathway as a necessary mechanism for transcriptional activity of the IL-6 promoter (12Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar). Our present data with the nuclear fusion protein Gal4-p65 show that the basal constitutive transcriptional activity of NF-κB p65, but not that of another acidic transactivator like VP16, could be specifically enhanced by TNF, independently of effects involving the cytoplasmic activation of NF-κB. This increased transcriptional activity is the result of the activation of MAPK pathways by TNF. The p38 as well as the ERK MAPK pathway contribute to the specific up-regulation by TNF without affecting the basal TNF-independent activity. This suggests a signalization system of at least two steps from the TNF receptor to the gene, in which, first, NF-κB becomes activated in the cytoplasm by a IκBα-specific kinase, as recently reported (37Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar), and second, the “nuclear” transactivation potential of the DNA-bound complex is modulated by (an) additional phosphorylation event(s) via different TNF-activated MAPKs. Such a multilevel regulation allows fine tuning and/or gene specific modulation of the transcriptional activity.Whether MAPKs act directly on the p65 transcription activation complex, or via intermediate kinases, remains an open question. Nevertheless, byin vitro kinase assays it was shown that neither IκBα, nor the NF-κB DNA-binding subunits p50, nor the transactivating C-terminal half of p65 became phosphorylated by p38. However, it is still conceivable that, in vivo, a kinase downstream in the p38 and ERK pathways phosphorylates NF-κB subunits (38Wesselborg S. Bauer M.K.A. Vogt M. Schmitz M.L. Schulze-Osthoff K. J. Biol. Chem. 1997; 272: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar).Furthermore, whether MAPK-dependent phosphorylation effectively takes place on the p65 subunit itself is also not proven. Evidence for phosphorylation-dependent regulation of NF-κB has already been reported by Naumann and Scheidereit (39Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar), who found increased binding of NF-κB upon phosphorylation of the p65 subunit. Recently, Zhong and co-workers (40Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar) showed a strongly increased transcriptional activity after phosphorylation of p65 on a consensus cAMP-dependent protein kinase site, which is located in the p65 Rel homology domain. However, since this site is clearly different and distinct from the p65 transactivation domains TA1 and TA2, our data point to another phosphorylation system. Schmitzet al. (21Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) also observed increased transcriptional activity upon treatment of HeLa cells with phorbol ester and suggested a possible phosphorylation in the p65 TA2 domain by a protein kinase C-dependent mechanism. It cannot, however, be excluded that different signals and/or stimuli converge into the same activation region of the p65 subunit.Recent data connect transcriptional activity of the κB p65 subunit with the versatile coactivator/cointegrator proteins p300 and CBP (41Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (710) Google Scholar,42Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). Extensive protein-protein interactions have been mapped between the N- and C-terminal regions of CBP/p300, and the C terminus of p65, containing both transactivation domains. Interestingly, since our results with the Gal4-p65 fusion proteins demonstrate a crucial role of these domains of p65 for TNF inducibility, the possible phosphorylation status of these domains in p65-CBP interaction may be of particular interest. Furthermore, the coactivator proteins CBP/p300 are subject themselves to phosphorylation control and were described as a nuclear target for S6 kinase pp90rsk and for cyclin-dependent kinases (43Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar). Other targets for MAPK phosphorylation are part of the RNA polymerase complex (28Bellier S. Dubois M.-F. Nishida E. Almouzni G. Bensaude O. Mol. Cell. Biol. 1997; 17: 1434-1440Crossref PubMed Scopus (54) Google Scholar, 44Venetianer A. Dubois M.F. Nguyen V.T. Bellier S. Seo S.J. Bensaude O. Eur. J. Biochem. 1995; 233: 83-92Crossref PubMed Scopus (42) Google Scholar, 45Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar). Since RNA polymerase II is constitutively associated with CBP/p300, interaction of the coactivator with NF-κB in the IL-6 promoter complex may efficiently recruit the polymerase complex, to trigger subsequent IL-6 gene expression (46Kee B.L. Arias J. Montminy M.R. J. Biol. Chem. 1996; 271: 2373-2375Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar).In summary, our data show that p38 and ERK MAPK signaling pathways constitute an additional level of gene regulation by the transcription factor NF-κB, more particularly of the p65 subunit, in response to TNF. Modulation of the p65 transactivation occurs in the nucleus and independent from IκB regulation, since it is faithfully reproduced with the Gal4 fusion proteins. However, whether in vivo the κB p65 subunit itself is a direct or indirect substrate of TNF-activated p38 and/or ERK MAPK pathways, or else is part of an integrated transcriptionally active complex, which is subject to modulation by MAPK phosphorylation, needs further study. Interleukin (IL) 1The abbreviations used are: IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; CBP, cAMP responsive element binding protein binding protein; Tricine, N-tris(hydroxymethyl)methylglycine. 1The abbreviations used are: IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; CBP, cAMP responsive element binding protein binding protein; Tricine, N-tris(hydroxymethyl)methylglycine. 6 contributes to a multitude of physiological and pathophysiological processes. Among its many functions, IL-6 plays an active role in immunological responses, bone metabolism, reproduction, inflammation, neoplasia, and aging. The cellular and molecular biology of IL-6 has been explored by a variety of approaches (1Hirano T. Kishimoto T. Res. Immunol. 1992; 143: 689-788Google Scholar, 2Keller E.T. Wanagat J. Ershler W.B. Frontiers Biosci. 1996; 1: d340-d357Crossref PubMed Scopus (124) Google Scholar). The regulation of expression of the IL-6 gene is adapted to the key function of this cytokine, namely a systemic alarm signal that recruits diverse host defense mechanisms that serve to limit tissue injury. Inflammation-associated cytokines such as tumor necrosis factor (TNF), IL-1, and platelet-derived growth factor, bacterial products such as endotoxin, and acute viral infections, all enhance IL-6 gene expression. The characterization of the IL-6 promoter revealed a complex control region that can be triggered by multiple activation pathways (3Dendorfer U. Oettgen P. Libermann T.A. Mol. Cell. Biol. 1994; 14: 4443-4454Crossref PubMed Scopus (298) Google Scholar, 4Haegeman G. Fiers W. Packer L. Wirtz K. Signalling Mechanisms: From Transcription Factors to Oxidative Stress. Springer Verlag, Berlin1995: 375-382Google Scholar). In the case of TNF (and of IL-1), the main transcriptional activator for IL-6 gene induction is the nuclear factor (NF)-κB (5Libermann T.A. Baltimore D. Mol. Cell. Biol. 1990; 10: 2327-2334Crossref PubMed Google Scholar, 6Shimizu H. Mitomo K. Watanabe T. Okamoto S. Yamamoto K. Mol. Cell. Biol. 1990; 10: 561-568Crossref PubMed Scopus (316) Google Scholar, 7Zhang Y. Lin J.-X. Vilček J. Mol. Cell. Biol. 1990; 10: 3818-3823Crossref PubMed Scopus (233) Google Scholar), which is typically a dimer between p50 and the transactivating subunit p65 (RelA). In unstimulated cells, NF-κB resides in the cytoplasm. Here, the DNA-binding dimer is bound to the inhibitory molecule IκB, from which it is released upon cell stimulation. NF-κB then migrates into the nucleus, where it effects expression of its numerous target genes (8Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4582) Google Scholar). We have shown previously that in the mouse fibrosarcoma cell line L929sA, the quantities of activated NF-κB do not correlate with the variations of IL-6 mRNA levels in the cell. We therefore concluded that other cooperative factors or regulatory mechanisms are necessary for modulating the levels of NF-κB-dependent IL-6 mRNA production (9Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar). Studies over the last few years have shown that different mitogen-activated kinase (MAPK) cascade pathways contribute to the transmission of extracellular signals that can finally result in direct or indirect phosphorylation of various transcription factors and alterations in gene expression (10Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1160) Google Scholar, 11Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). More particularly, we reported that abrogation of the p38 MAPK pathway represses TNF-mediated IL-6 gene expression, but not NF-κB activation and DNA binding (12Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar). In the present article, we report on the essential role of NF-κB to trigger IL-6 gene activation in response to TNF in L929sA cells. Furthermore, we show that, apart from TNF-induced cytoplasmic NF-κB activation and nuclear DNA binding, the TNF-activated p38 and ERK MAPK pathways contribute to transcriptional activation by modulating the transactivation capacity of the NF-κB p65 subunit. DISCUSSIONIn this paper, we have focused on the transcriptional activation by the factor NF-κB in response to TNF. By the use of various deleted and point-mutated versions of the IL-6 promoter, we have documented the key role of the κB motif for induction by TNF. A number of previous reports have already described the necessity of cooperation and association of NF-κB with other DNA-bound transcription factors for optimal gene activation (33Kaszubska W. Hooft van Huijsduijnen R. Ghersa P. DeRaemy-Schenk A.-M. Chen B.P.C. Hai T. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1993; 13: 7180-7190Crossref PubMed Google Scholar, 34Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (873) Google Scholar, 35Stein B. Baldwin Jr., A.S. Ballard D.W. Greene W.C. Angel P. Herrlich P. EMBO J. 1993; 12: 3879-3891Crossref PubMed Scopus (567) Google Scholar, 36Ray A. Hannink M. Ray B.K. J. Biol. Chem. 1995; 270: 7365-7374Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The deletion analysis of the IL-6 promoter shows that such factors have indeed a co-activating and integrating function for full stimulation of the IL-6 promoter. However, using “loss-of-function” mutants of the IL-6 promoter, the crucial role of NF-κB is obvious, without the primary need for other associating DNA-bound factors. Using the “gain-of-function” approach by inserting multiple κB sites in front of an unresponsive promoter, the TNF response could be restored.Activation of NF-κB and its binding to DNA is, however, not sufficient for IL-6 gene activation by TNF; the requirement of additional activating mechanisms has already been described previously (4Haegeman G. Fiers W. Packer L. Wirtz K. Signalling Mechanisms: From Transcription Factors to Oxidative Stress. Springer Verlag, Berlin1995: 375-382Google Scholar, 9Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar). More particularly, recently, we have established the importance of the p38 MAPK pathway as a necessary mechanism for transcriptional activity of the IL-6 promoter (12Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar). Our present data with the nuclear fusion protein Gal4-p65 show that the basal constitutive transcriptional activity of NF-κB p65, but not that of another acidic transactivator like VP16, could be specifically enhanced by TNF, independently of effects involving the cytoplasmic activation of NF-κB. This increased transcriptional activity is the result of the activation of MAPK pathways by TNF. The p38 as well as the ERK MAPK pathway contribute to the specific up-regulation by TNF without affecting the basal TNF-independent activity. This suggests a signalization system of at least two steps from the TNF receptor to the gene, in which, first, NF-κB becomes activated in the cytoplasm by a IκBα-specific kinase, as recently reported (37Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar), and second, the “nuclear” transactivation potential of the DNA-bound complex is modulated by (an) additional phosphorylation event(s) via different TNF-activated MAPKs. Such a multilevel regulation allows fine tuning and/or gene specific modulation of the transcriptional activity.Whether MAPKs act directly on the p65 transcription activation complex, or via intermediate kinases, remains an open question. Nevertheless, byin vitro kinase assays it was shown that neither IκBα, nor the NF-κB DNA-binding subunits p50, nor the transactivating C-terminal half of p65 became phosphorylated by p38. However, it is still conceivable that, in vivo, a kinase downstream in the p38 and ERK pathways phosphorylates NF-κB subunits (38Wesselborg S. Bauer M.K.A. Vogt M. Schmitz M.L. Schulze-Osthoff K. J. Biol. Chem. 1997; 272: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar).Furthermore, whether MAPK-dependent phosphorylation effectively takes place on the p65 subunit itself is also not proven. Evidence for phosphorylation-dependent regulation of NF-κB has already been reported by Naumann and Scheidereit (39Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar), who found increased binding of NF-κB upon phosphorylation of the p65 subunit. Recently, Zhong and co-workers (40Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar) showed a strongly increased transcriptional activity after phosphorylation of p65 on a consensus cAMP-dependent protein kinase site, which is located in the p65 Rel homology domain. However, since this site is clearly different and distinct from the p65 transactivation domains TA1 and TA2, our data point to another phosphorylation system. Schmitzet al. (21Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) also observed increased transcriptional activity upon treatment of HeLa cells with phorbol ester and suggested a possible phosphorylation in the p65 TA2 domain by a protein kinase C-dependent mechanism. It cannot, however, be excluded that different signals and/or stimuli converge into the same activation region of the p65 subunit.Recent data connect transcriptional activity of the κB p65 subunit with the versatile coactivator/cointegrator proteins p300 and CBP (41Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (710) Google Scholar,42Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). Extensive protein-protein interactions have been mapped between the N- and C-terminal regions of CBP/p300, and the C terminus of p65, containing both transactivation domains. Interestingly, since our results with the Gal4-p65 fusion proteins demonstrate a crucial role of these domains of p65 for TNF inducibility, the possible phosphorylation status of these domains in p65-CBP interaction may be of particular interest. Furthermore, the coactivator proteins CBP/p300 are subject themselves to phosphorylation control and were described as a nuclear target for S6 kinase pp90rsk and for cyclin-dependent kinases (43Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar). Other targets for MAPK phosphorylation are part of the RNA polymerase complex (28Bellier S. Dubois M.-F. Nishida E. Almouzni G. Bensaude O. Mol. Cell. Biol. 1997; 17: 1434-1440Crossref PubMed Scopus (54) Google Scholar, 44Venetianer A. Dubois M.F. Nguyen V.T. Bellier S. Seo S.J. Bensaude O. Eur. J. Biochem. 1995; 233: 83-92Crossref PubMed Scopus (42) Google Scholar, 45Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar). Since RNA polymerase II is constitutively associated with CBP/p300, interaction of the coactivator with NF-κB in the IL-6 promoter complex may efficiently recruit the polymerase complex, to trigger subsequent IL-6 gene expression (46Kee B.L. Arias J. Montminy M.R. J. Biol. Chem. 1996; 271: 2373-2375Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar).In summary, our data show that p38 and ERK MAPK signaling pathways constitute an additional level of gene regulation by the transcription factor NF-κB, more particularly of the p65 subunit, in response to TNF. Modulation of the p65 transactivation occurs in the nucleus and independent from IκB regulation, since it is faithfully reproduced with the Gal4 fusion proteins. However, whether in vivo the κB p65 subunit itself is a direct or indirect substrate of TNF-activated p38 and/or ERK MAPK pathways, or else is part of an integrated transcriptionally active complex, which is subject to modulation by MAPK phosphorylation, needs further study. In this paper, we have focused on the transcriptional activation by the factor NF-κB in response to TNF. By the use of various deleted and point-mutated versions of the IL-6 promoter, we have documented the key role of the κB motif for induction by TNF. A number of previous reports have already described the necessity of cooperation and association of NF-κB with other DNA-bound transcription factors for optimal gene activation (33Kaszubska W. Hooft van Huijsduijnen R. Ghersa P. DeRaemy-Schenk A.-M. Chen B.P.C. Hai T. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1993; 13: 7180-7190Crossref PubMed Google Scholar, 34Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (873) Google Scholar, 35Stein B. Baldwin Jr., A.S. Ballard D.W. Greene W.C. Angel P. Herrlich P. EMBO J. 1993; 12: 3879-3891Crossref PubMed Scopus (567) Google Scholar, 36Ray A. Hannink M. Ray B.K. J. Biol. Chem. 1995; 270: 7365-7374Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The deletion analysis of the IL-6 promoter shows that such factors have indeed a co-activating and integrating function for full stimulation of the IL-6 promoter. However, using “loss-of-function” mutants of the IL-6 promoter, the crucial role of NF-κB is obvious, without the primary need for other associating DNA-bound factors. Using the “gain-of-function” approach by inserting multiple κB sites in front of an unresponsive promoter, the TNF response could be restored. Activation of NF-κB and its binding to DNA is, however, not sufficient for IL-6 gene activation by TNF; the requirement of additional activating mechanisms has already been described previously (4Haegeman G. Fiers W. Packer L. Wirtz K. Signalling Mechanisms: From Transcription Factors to Oxidative Stress. Springer Verlag, Berlin1995: 375-382Google Scholar, 9Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar). More particularly, recently, we have established the importance of the p38 MAPK pathway as a necessary mechanism for transcriptional activity of the IL-6 promoter (12Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar). Our present data with the nuclear fusion protein Gal4-p65 show that the basal constitutive transcriptional activity of NF-κB p65, but not that of another acidic transactivator like VP16, could be specifically enhanced by TNF, independently of effects involving the cytoplasmic activation of NF-κB. This increased transcriptional activity is the result of the activation of MAPK pathways by TNF. The p38 as well as the ERK MAPK pathway contribute to the specific up-regulation by TNF without affecting the basal TNF-independent activity. This suggests a signalization system of at least two steps from the TNF receptor to the gene, in which, first, NF-κB becomes activated in the cytoplasm by a IκBα-specific kinase, as recently reported (37Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar), and second, the “nuclear” transactivation potential of the DNA-bound complex is modulated by (an) additional phosphorylation event(s) via different TNF-activated MAPKs. Such a multilevel regulation allows fine tuning and/or gene specific modulation of the transcriptional activity. Whether MAPKs act directly on the p65 transcription activation complex, or via intermediate kinases, remains an open question. Nevertheless, byin vitro kinase assays it was shown that neither IκBα, nor the NF-κB DNA-binding subunits p50, nor the transactivating C-terminal half of p65 became phosphorylated by p38. However, it is still conceivable that, in vivo, a kinase downstream in the p38 and ERK pathways phosphorylates NF-κB subunits (38Wesselborg S. Bauer M.K.A. Vogt M. Schmitz M.L. Schulze-Osthoff K. J. Biol. Chem. 1997; 272: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Furthermore, whether MAPK-dependent phosphorylation effectively takes place on the p65 subunit itself is also not proven. Evidence for phosphorylation-dependent regulation of NF-κB has already been reported by Naumann and Scheidereit (39Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar), who found increased binding of NF-κB upon phosphorylation of the p65 subunit. Recently, Zhong and co-workers (40Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar) showed a strongly increased transcriptional activity after phosphorylation of p65 on a consensus cAMP-dependent protein kinase site, which is located in the p65 Rel homology domain. However, since this site is clearly different and distinct from the p65 transactivation domains TA1 and TA2, our data point to another phosphorylation system. Schmitzet al. (21Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) also observed increased transcriptional activity upon treatment of HeLa cells with phorbol ester and suggested a possible phosphorylation in the p65 TA2 domain by a protein kinase C-dependent mechanism. It cannot, however, be excluded that different signals and/or stimuli converge into the same activation region of the p65 subunit. Recent data connect transcriptional activity of the κB p65 subunit with the versatile coactivator/cointegrator proteins p300 and CBP (41Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (710) Google Scholar,42Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). Extensive protein-protein interactions have been mapped between the N- and C-terminal regions of CBP/p300, and the C terminus of p65, containing both transactivation domains. Interestingly, since our results with the Gal4-p65 fusion proteins demonstrate a crucial role of these domains of p65 for TNF inducibility, the possible phosphorylation status of these domains in p65-CBP interaction may be of particular interest. Furthermore, the coactivator proteins CBP/p300 are subject themselves to phosphorylation control and were described as a nuclear target for S6 kinase pp90rsk and for cyclin-dependent kinases (43Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar). Other targets for MAPK phosphorylation are part of the RNA polymerase complex (28Bellier S. Dubois M.-F. Nishida E. Almouzni G. Bensaude O. Mol. Cell. Biol. 1997; 17: 1434-1440Crossref PubMed Scopus (54) Google Scholar, 44Venetianer A. Dubois M.F. Nguyen V.T. Bellier S. Seo S.J. Bensaude O. Eur. J. Biochem. 1995; 233: 83-92Crossref PubMed Scopus (42) Google Scholar, 45Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar). Since RNA polymerase II is constitutively associated with CBP/p300, interaction of the coactivator with NF-κB in the IL-6 promoter complex may efficiently recruit the polymerase complex, to trigger subsequent IL-6 gene expression (46Kee B.L. Arias J. Montminy M.R. J. Biol. Chem. 1996; 271: 2373-2375Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In summary, our data show that p38 and ERK MAPK signaling pathways constitute an additional level of gene regulation by the transcription factor NF-κB, more particularly of the p65 subunit, in response to TNF. Modulation of the p65 transactivation occurs in the nucleus and independent from IκB regulation, since it is faithfully reproduced with the Gal4 fusion proteins. However, whether in vivo the κB p65 subunit itself is a direct or indirect substrate of TNF-activated p38 and/or ERK MAPK pathways, or else is part of an integrated transcriptionally active complex, which is subject to modulation by MAPK phosphorylation, needs further study. We thank Drs. J. Lee and P. Cohen for providing SB203580, and Dr. P. Soriano for donating pPGKβGeobpA. We acknowledge F. Cherbal, I. Van Rompaey, and F. Molemans for technical assistance." @default.
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