Transcriptional Regulation of EGR-1 by the Interleukin-1-JNK-MKK7-c-Jun Pathway

Abstract

The proinflammatory cytokine interleukin (IL)-1 activates several hundred genes within the same cell. This occurs in part by activation of the MKK7-JNK-c-Jun signaling pathway whose precise role in the regulation of individual inflammatory genes is still incompletely understood. To identify the genes that are under specific control of activated JNK, we used a JNK-MKK7 fusion protein. Genome-wide microarray analysis revealed EGR-1 as the transcript that was most strongly induced by JNK-MKK7. IL-1-stimulated EGR-1 mRNA and protein expression were impaired in cells lacking JNK or c-Jun. Transcriptional activation of the EGR-1 promoter by JNK-MKK7 or by IL-1 required a single upstream AP-1 site and three distal serum-response elements (SRE). Reconstitution experiments in c-Jun-deficient cells revealed that c-Jun is required for EGR-1 transcription through both the AP-1 site and the distal SREs. By chromatin immunoprecipitation analysis, we found IL-1-inducible recruitment of c-Jun to the AP-1 site and to the region containing the three distal SREs. These experiments suggest that c-Jun plays a dual role in EGR-1 transcription. It directly binds to the AP-1 element, and at the same time it is essential for promoter activation through the three distal SREs by an indirect unknown mechanism. As predicted by TRANSFAC analysis and verified by ChIP experiments, IL-1-induced EGR-1 protein binds to the promoter regions of inflammatory mediators such as IL-6, IL-8, and CCL2. Furthermore, short interfering RNA-mediated suppression of EGR-1 partially suppresses IL-1-inducible transcription of IL-8, IL-6, and CCL2. In summary, we provide novel evidence for a complex c-Jun-mediated mechanism that is essential for inducible EGR-1 expression. We identify this pathway as a previously unrecognized part of a multistep gene regulatory network that controls cytokine and chemokine expression via the IL-1-MKK7-JNK-c-Jun-EGR-1 pathway. The proinflammatory cytokine interleukin (IL)-1 activates several hundred genes within the same cell. This occurs in part by activation of the MKK7-JNK-c-Jun signaling pathway whose precise role in the regulation of individual inflammatory genes is still incompletely understood. To identify the genes that are under specific control of activated JNK, we used a JNK-MKK7 fusion protein. Genome-wide microarray analysis revealed EGR-1 as the transcript that was most strongly induced by JNK-MKK7. IL-1-stimulated EGR-1 mRNA and protein expression were impaired in cells lacking JNK or c-Jun. Transcriptional activation of the EGR-1 promoter by JNK-MKK7 or by IL-1 required a single upstream AP-1 site and three distal serum-response elements (SRE). Reconstitution experiments in c-Jun-deficient cells revealed that c-Jun is required for EGR-1 transcription through both the AP-1 site and the distal SREs. By chromatin immunoprecipitation analysis, we found IL-1-inducible recruitment of c-Jun to the AP-1 site and to the region containing the three distal SREs. These experiments suggest that c-Jun plays a dual role in EGR-1 transcription. It directly binds to the AP-1 element, and at the same time it is essential for promoter activation through the three distal SREs by an indirect unknown mechanism. As predicted by TRANSFAC analysis and verified by ChIP experiments, IL-1-induced EGR-1 protein binds to the promoter regions of inflammatory mediators such as IL-6, IL-8, and CCL2. Furthermore, short interfering RNA-mediated suppression of EGR-1 partially suppresses IL-1-inducible transcription of IL-8, IL-6, and CCL2. In summary, we provide novel evidence for a complex c-Jun-mediated mechanism that is essential for inducible EGR-1 expression. We identify this pathway as a previously unrecognized part of a multistep gene regulatory network that controls cytokine and chemokine expression via the IL-1-MKK7-JNK-c-Jun-EGR-1 pathway. The inflammatory response is characterized by the coordinated induction or repression of a large number of inflammatory genes. These genes control leukocyte infiltration, innate or adaptive immune responses, and tissue remodeling. Prototypic proinflammatory cytokines such as interleukin (IL) 3The abbreviations used are: IL, interleukin; AP-1, activating protein-1; SRE, serum-response element; MAP, mitogen-activated protein; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; RNAi, RNA interference; siRNA, short interfering RNA; RT, reverse transcription; PBS, phosphate-buffered saline; TCF, ternary complex factor; TBS, Tris-buffered saline; Mef, murine embryonic fibroblast; kd, kinase dead. -1 or tumor necrosis factor (TNF) can activate or repress several hundred genes within the same cell (1Thiefes A. Wolter S. Mushinski J.F. Hoffmann E. Dittrich-Breiholz O. Graue N. Dorrie A. Schneider H. Wirth D. Luckow B. Resch K. Kracht M. J. Biol. Chem. 2005; 280: 27728-27741Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). They mediate their gene-regulatory effects in part by activation of MAPK signaling pathways such as JNK, p38, and ERK. These pathways in turn activate a large number of transcriptional regulators that fall into different classes, i.e. AP-1, ets, C/EBP, and others. Although it is well known that JNKs phosphorylate and activate AP-1 components such as c-Jun or ATF-2, the precise role of individual transcription factors in regulation of individual inflammatory genes is comparably less understood. Moreover, most inflammatory genes bind several transcription factors simultaneously, the mRNA expression of which is themselves regulated at the transcriptional level. For example, the c-JUN promoter is regulated by a c-Jun-ATF-2 dimer in an autoregulated fashion (2van Dam H. Duyndam M. Rottier R. Bosch A. Vries-Smits L. Herrlich P. Zantema A. Angel P. van der Eb A.J. EMBO J. 1993; 12: 479-487Crossref PubMed Scopus (342) Google Scholar, 3Angel P. Hattori K. Smeal T. Karin M. Cell. 1988; 55: 875-885Abstract Full Text PDF PubMed Scopus (993) Google Scholar). But it is also regulated by lipopolysaccharide-induced p38-mediated activation of the transcription factor Mef2c (4Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (685) Google Scholar). Hence, an emerging picture is that the initial inflammatory gene expression program is a result of complex mutual control of transcription factor networks and their target genes. A comprehensive understanding of these networks requires the identification of all target genes in a given pathway, a task that is still far from complete. This is exemplified by studies on the JNK-c-Jun pathway. Although JNKs are activated by almost every proinflammatory or stressful condition, their downstream target genes are poorly defined. This also applies to the downstream effector of JNK, c-Jun. One way of studying the role of JNK in gene regulation is selective activation of the pathway by ectopically expressed gain-of-function mutants. We have previously used an active mutant of MKK7, the only specific upstream activator of JNK (5Wolter S. Mushinski J.F. Saboori A.M. Resch K. Kracht M. J. Biol. Chem. 2002; 277: 3576-3584Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). However, this mutant is a relatively weak activator of JNK and may thus not mimic the strong increase of activity of endogenous JNK that is found in response to proinflammatory cytokines such as IL-1. Hence, we extended these studies using ectopically expressed fusion proteins of MKK7 and JNK. As shown previously, these fusion proteins display strong catalytic activity of JNK (6Rennefahrt U. Illert B. Greiner A. Rapp U.R. Troppmair J. Cancer Lett. 2004; 215: 113-124Crossref PubMed Scopus (10) Google Scholar, 7Rennefahrt U.E. Illert B. Kerkhoff E. Troppmair J. Rapp U.R. J. Biol. Chem. 2002; 277: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 8Zheng C. Xiang J. Hunter T. Lin A. J. Biol. 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Chem. 2005; 280: 27728-27741Abstract Full Text Full Text PDF PubMed Scopus (52) Google have A of the promoter to was from by to the promoter The was into the of using the as in H. R. P. S. Hoffmann E. D. Resch K. Kracht M. Cell. Biol. 1999; PubMed Scopus Google Scholar). was from and were from Angel and at well of were a of of by the c-Jun-deficient cells at in were of and of was in by of and gene activity was as previously (1Thiefes A. Wolter S. Mushinski J.F. Hoffmann E. Dittrich-Breiholz O. Graue N. Dorrie A. Schneider H. Wirth D. Luckow B. Resch K. Kracht M. J. Biol. Chem. 2005; 280: 27728-27741Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, H. R. P. S. Hoffmann E. D. Resch K. Kracht M. Cell. Biol. 1999; PubMed Scopus Google Scholar). EGR-1 siRNA, and control were from For experiments, cells were of and of cells were using were by for were used for murine promoter AP-1 and murine promoter distal three and and and and of RNA was as previously E. A. D. Dittrich-Breiholz O. Schneider H. Resch K. Kracht M. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google and into using murine reverse in a of of this was used to using on for murine or EGR-1 and murine or on an time The for individual was by the and for EGR-1 were by the for The were used to of mRNA expression as of mRNA expression of cells to the to of or of as in the were used for to were in in to of at this was by containing to the the was and the cells were and into were at at in PBS, and in and were by for on and at at for were and in at for of from or of control were to to of and the were at of a protein in was to the and was for at were by in of in in in at and in of were for at and of were to including A of the initial were to containing and A. at was and both and were for at at by at were to in and was using or to the was or and at was on and using of of and of of in a of were as at of by a at for were by and by using an and the of ChIP by from ChIP were by time using an The of ChIP or of and of the or in a of were as and analysis revealed a single of revealed that were from to and were used to of by the were in cell and or and as previously H. R. P. S. Hoffmann E. D. Resch K. Kracht M. Cell. Biol. 1999; PubMed Scopus Google Scholar). proteins were on and to in Tris-buffered were for in TBS, and for the in were by using the or was from cell and JNK activity was in by protein kinase using as previously (5Wolter S. Mushinski J.F. Saboori A.M. Resch K. Kracht M. J. Biol. Chem. 2002; 277: 3576-3584Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). RNA from as in the of was the including of RNA were used to the to the and were by an of were to the and were as by the in the of EGR-1 as JNK-MKK7 target gene by mRNA expression of on of used for protein kinase kinase growth response necrosis factor member growth response growth factor factor necrosis factor member family member containing response factor pathway containing transcription factor and protein growth factor in a and were on an at different to the of the was the were into by a of was the to the all of were to of and of the were is the of of the for all of a and is the of a of a the of or were and from of mRNA expression were in by the of the by the of the control To identify genes that are under control of activated JNK, we used a previously characterized JNK-MKK7 fusion protein. As shown expression of this not of a in of JNK by the MKK7 and a strong increase in catalytic activity of JNK as by of c-Jun in this not activate endogenous JNKs as shown previously U.E. Illert B. Kerkhoff E. Troppmair J. Rapp U.R. J. Biol. Chem. 2002; 277: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A for this is that the JNK-MKK7 fusion endogenous and upstream of the endogenous JNK pathway R.J. Biol. PubMed Scopus Google such as A.J. J. C. J. R.J. Science. PubMed Scopus Google or Lin R.J. R.J. Science. 2000; PubMed Google Scholar). However, it the of c-Jun and strongly endogenous c-Jun Hence, the JNK-MKK7 fusion protein a specific to the of c-Jun in the regulation of gene expression. we a analysis of mRNA expression in response to JNK-MKK7. As shown in we found genes by JNK-MKK7 by at a number of genes also required the catalytic activity of JNK-MKK7 that of JNK-MKK7. EGR-1 mRNA expression was by and was by far the most strongly transcript induced by JNK-MKK7. induction was almost on active JNK as was the only microarray study JNK-MKK7 fusion proteins S.Y. Kim S.H. Heasley L.E. J. Biol. Chem. 2002; 277: 47167-47174Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). to this EGR-1 was not as a JNK response and was the genes that were or S.Y. Kim S.H. Heasley L.E. J. Biol. Chem. 2002; 277: 47167-47174Abstract Full Text Full Text PDF PubMed Scopus (49) Google in The for this different JNK fusion different cell or different types used as We the of EGR-1 by JNK-MKK7 at the mRNA using EGR-1 is an stress-induced transcription the of regulation by stress-induced signaling pathways are poorly Hence, we regulation by the JNK-c-Jun pathway in murine In IL-1 induced a increase in mRNA In cells lacking mRNA induction by IL-1 was at and it was in cells lacking c-Jun or a mutant In to the EGR-1 protein was induced by IL-1 in This was as by the also by the JNK in c-Jun-deficient cells These that induction of expression by proinflammatory is under control of the JNK-c-Jun pathway. Moreover, to not previously as a c-Jun target including studies in which c-Jun-deficient were by a microarray for genes L. L. S. M. S. M. P. A. Angel P. 2004; PubMed Scopus Google Scholar). The EGR-1 promoter characterized in In SREs that bind serum-response factor and ternary complex factors such as or have in several are and activated by JNK, ERK, and Nat. Biol. PubMed Scopus Google Scholar, A.J. P. R.J. Science. PubMed Scopus Google Scholar, A.J. S.H. R.J. Cell. Biol. 1997; PubMed Scopus Google Scholar, S.H. A.J. R.J. Cell. Biol. PubMed Scopus Google Scholar, S.H. A.J. R.J. EMBO J. PubMed Scopus Google Scholar, R. Hunter T. EMBO J. 1997; PubMed Scopus Google Scholar, R. Hunter T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google shown that JNK R. Kracht M. C. A. Cell. Biol. PubMed Scopus Google Scholar). We used a of the murine promoter and mutant as in M. R. A. P. S.F. Stern D. Mackman N. PubMed Scopus Google to to the by JNK-MKK7 The active not the induced transcription by A mutant lacking an upstream AP-1 site was only induced by and lacking the three distal SREs the AP-1 site to JNK-MKK7 IL-1 induced promoter activation required both the AP-1 site and the three distal SREs in an fashion In the distal AP-1 site was also required for promoter activity as in activity and Hence, the promoter from the JNK pathway through the distal AP-1 site and three of the previously characterized SREs. This result that the JNK-MKK7 pathway activate transcription by a dual c-Jun-mediated activation via the AP-1 site and and activation of proteins such as or a role for c-Jun in mRNA expression not previously, we role in transcription in using murine lacking or cells that were c-Jun expression of promoter in response to specific activation by JNK-MKK7 was almost in c-Jun-deficient cells or in cells a c-Jun mutant lacking the However, transcription in response to was in cells a c-Jun expression the essential role of c-Jun in transcription in to cells and activity of the promoter lacking the AP-1 site was in murine cells This that the distal AP-1 site not only to inducible transcription also controls mRNA in a cell transcription requires c-Jun. c-Jun-deficient were a c-Jun expression were JNK-MKK7 kd, or expression of JNK-MKK7 or c-Jun was by analysis of cell using or that c-Jun on a protein that was used to control for protein c-Jun-deficient were c-Jun or a c-Jun mutant lacking the were JNK-MKK7 kd, or in to promoter both the AP-1 and or lacking the AP-1 site as cells were and activity was are the from activation of the promoter lacking the AP-1 site was also on c-Jun Hence, the experiments shown in that c-Jun plays a dual role in transcription. By directly to the AP-1 it a from the activated pathway. the same time it is essential for promoter activation through the three distal SREs. By V. E. S. A. D. M. K. N. P. B. H. E. Res. PubMed Scopus Google analysis we not AP-1 within the upstream of the transcriptional site Hence, JNK transcription via c-Jun through the SREs by an indirect mechanism. This was by the of c-Jun chromatin the AP-1 site or the three SREs using of from ChIP experiments were by using time to for in We found IL-1-induced specific and recruitment of c-Jun to both the region the AP-1 site and to the region containing the three distal SREs of chromatin by c-Jun was specific ChIP from c-Jun-deficient cells or by using control In to c-Jun ChIP strong the three SREs as and of to the AP-1 site protein expression was in and not In summary, to the experiments shown in and c-Jun is directly to a single AP-1 site and to the three SREs to transcription. This indirect mechanism may such as or or to identify components of the It was shown previously that is upstream of EGR-1 in the signaling pathway M. R. A. P. S.F. Stern D. Mackman N. PubMed Scopus Google Scholar). study this transcription factor regulatory network by that c-Jun is in EGR-1 transcriptional Hence, and EGR-1 a transcription factor The mutual control of these immediate early gene transcription factor networks required for strong transcription of proinflammatory of EGR-1 in or suppression by in cells shown to result in expression of IL-8, and in response to and S.F. Fujita T. Lu J. Okada K. Shan Z.Y. Mackman N. Pinsky D.J. Stern D.M. Nat. Med. 2000; 6: 1355-1361Crossref PubMed Scopus (398) Google Scholar, R. B. B. M. Mackman N. PubMed Scopus Google Scholar, J. A. H. D. J. J. Collins T. J.M. J. Med. PubMed Scopus Google Scholar, J. Immunol. PubMed Scopus Google Scholar). We the upstream regions of of these genes for EGR-1 and as both factors elements A. J. Cell. 2005; PubMed Scopus Google Scholar, L.M. Williams A.J. Collins T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We found a large number of i.e. or for and were predicted by V. E. S. A. D. M. K. N. P. B. H. E. Res. PubMed Scopus Google Scholar, E. P. H. R. Res. 1996; PubMed Scopus Google using the in To these we regulatory regions of all three genes that were previously shown to to IL-1 or These regions also whose for inducible regulation of IL-6, and by studies E. Dittrich-Breiholz O. H. Kracht M. J. Biol. 2002; PubMed Google Scholar, Z. J.M. J. Immunol. PubMed Scopus Google Scholar, T. K. Y. N. K. T. S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). As shown in in to of these regions we found at predicted of endogenous EGR-1 to these was by ChIP analysis Moreover, EGR-1 partially IL-1-inducible activation of genes containing the regulatory regions of IL-8, IL-6, and a of EGR-1 for IL-1-inducible transcription suppression of EGR-1 transcriptional regulation of IL-8, IL-6, and CCL2. cells were EGR-1 or control cells were or were of EGR-1 mRNA was by time and is to cells the cells were as in A. of of promoter or of promoter were as cells cells were for or were cells were and activity in cell was and for is the activity from three experiments in cells were of of promoter or of promoter and control or EGR-1 as gene activity was as in B. is the activity from three experiments in In summary, we provide novel evidence for a complex c-Jun-mediated mechanism that EGR-1 mRNA expression. identify this pathway as an unrecognized part of a multistep gene regulatory network that controls cytokine and chemokine expression via the IL-1-MKK7-JNK-c-Jun-EGR-1 pathway These as a target for the of inflammatory through of this novel pathway.