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A Constitutive Active MEK → ERK Pathway Negatively Regulates NF-κB-dependent Gene Expression by Modulating TATA-binding Protein Phosphorylation

2000; Elsevier BV; Volume: 275; Issue: 36 Linguagem: Inglês

10.1074/jbc.m003599200

ISSN

1083-351X

Autores

A. Brent Carter, Gary W. Hunninghake,

Tópico(s)

Melanoma and MAPK Pathways

Resumo

Endotoxin-induced cytokine gene expression is regulated, in part, by NF-κB. We have shown that both the ERK and p38 mitogen-activated protein (MAP) kinases are necessary for cytokine gene transcription and that the p38 MAP kinase is required for NF-κB-driven transcription, so we hypothesized that the MEK → ERK pathway regulated NF-κB-driven transcription as well. We found that a constitutive active MEK → ERK pathway inhibited NF-κB-driven transcription. In addition, both PD 98059 and a dominant negative ERK2 augmented NF-κB-driven transcription; however, neither PD 98059 nor MEK1 altered NF-κB activation at any level. The constitutive active MEK → ERK pathway inhibited the phosphorylation of TBP, which is necessary for both interaction with RelA and binding to the TATA box. Due to the fact that we have shown that the p38 MAP kinase modulates TBP activation, we evaluated the effect of the constitutive active MEK → ERK pathway on p38 MAP kinase activity. We found that the MEK → ERK pathway negatively regulates NF-κB-driven transcription, in part, by inhibiting p38 MAP kinase activity. Thus, the ERK and p38 MAP kinases have differential effects on NF-κB-driven transcription. Endotoxin-induced cytokine gene expression is regulated, in part, by NF-κB. We have shown that both the ERK and p38 mitogen-activated protein (MAP) kinases are necessary for cytokine gene transcription and that the p38 MAP kinase is required for NF-κB-driven transcription, so we hypothesized that the MEK → ERK pathway regulated NF-κB-driven transcription as well. We found that a constitutive active MEK → ERK pathway inhibited NF-κB-driven transcription. In addition, both PD 98059 and a dominant negative ERK2 augmented NF-κB-driven transcription; however, neither PD 98059 nor MEK1 altered NF-κB activation at any level. The constitutive active MEK → ERK pathway inhibited the phosphorylation of TBP, which is necessary for both interaction with RelA and binding to the TATA box. Due to the fact that we have shown that the p38 MAP kinase modulates TBP activation, we evaluated the effect of the constitutive active MEK → ERK pathway on p38 MAP kinase activity. We found that the MEK → ERK pathway negatively regulates NF-κB-driven transcription, in part, by inhibiting p38 MAP kinase activity. Thus, the ERK and p38 MAP kinases have differential effects on NF-κB-driven transcription. nuclear factor κB IκB kinase transcription factor IIB TATA-binding protein transcription factor IID mitogen-activated protein extracellular signal-regulated kinase c-Jun NH2-terminal MAP kinase MAP kinase kinase MAP kinase kinase kinase 1 activating protein-1 CCAAT-enhancer binding protein β activating transcription factor-2 interleukin lipopolysaccharide chloramphenicol acetyltransferase polyacrylamide gel electrophoresis tumor necrosis factor A transcription factor that is necessary for the transcription of many genes involved in inflammatory and immune responses is nuclear factor κB (NF-κB)1 (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4641) Google Scholar,2Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1221) Google Scholar). NF-κB is a dimeric protein that is composed of members of the Rel family of transcription factors. The regulation of NF-κB-dependent gene expression can occur at multiple levels after cell stimulation. NF-κB is held in the cytoplasm by an inhibitor protein, IκB, in quiescent cells (3Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 4Matthews J.R. Hay R.T. Int. J. Biochem. Cell Biol. 1995; 27: 865-879Crossref PubMed Scopus (72) Google Scholar). In stimulated cells, the phosphorylation of IκBα on Ser-32 and Ser-36 and IκBβ on Ser-19 and Ser-23 by IKKα and IKKβ, respectively, results in their subsequent degradation (3Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 5Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5645) Google Scholar, 6Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. 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Transcriptional activity, however, also requires the phosphorylation of the transcriptionally active NF-κB subunits, RelA (p65), c-Rel, and RelB. In fact, several studies have shown that the phosphorylation of p65 is necessary for transcription to be initiated (12Hayashi T. Sekine T. Okamoto T. J. Biol. Chem. 1993; 268: 26790-26795Abstract Full Text PDF PubMed Google Scholar, 13Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (329) Google Scholar, 14Schmitz 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, 15Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 16Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar). The interaction of the p65 subunit with the basal transcription factors IIB (TFIIB) and TATA-binding protein (TBP) is also necessary for NF-κB-driven transcription (17Kerr L.D. Ransone L.J. Wamsley P. Schmitt M.J. Boyer T.G. Zhou Q. Berk A.J. Verma I.M. Nature. 1993; 365: 412-419Crossref PubMed Scopus (134) Google Scholar, 18Xu X. Prorock C. Ishikawa H. Maldonado E. Ito Y. Gelinas C. Mol. Cell. Biol. 1993; 13: 6733-6741Crossref PubMed Scopus (98) Google Scholar, 19Blair W.S. Bogerd H.P. Madore S.J. Cullen B.R. Mol. Cell. Biol. 1994; 14: 7226-7234Crossref PubMed Scopus (104) Google Scholar, 20Schmitz M.L. Stelzer G. Altmann H. Meisterernst M. Baeuerle P.A. J. Biol. Chem. 1995; 270: 7219-7226Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The phosphorylation of TBP is required for both its interaction with the p65 subunit and its binding to the TATA box (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 22Chibazakura T. Watanabe F. Kitajima S. Tsukada K. Yasukochi Y. Teraoka H. Eur. J. Biochem. 1997; 247: 1166-1173Crossref PubMed Scopus (41) Google Scholar). The mitogen-activated protein (MAP) kinases are a family of second messenger kinases that is essential for transferring signals from the cell surface to the nucleus. We have shown that the p38 MAP kinase regulates NF-κB-dependent gene expression by modulating the phosphorylation and subsequent activation of TBP (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The regulation of NF-κB-dependent transcription is important due to the fact that most cytokine promoter sequences have active NF-κB sites and thus suggests the mechanism by which the p38 MAP kinase controls cytokine gene expression. Other MAP kinases have been linked to cytokine gene expression. In addition to others (23Reimann T. Buscher D. Hipskind R.A. Krautwald S. Lohmann-Matthes M.L. Baccarini M. J. Immunol. 1994; 153: 5740-5749PubMed Google Scholar, 24Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell. Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar), we have shown that activation of the ERK kinase is important for the induction of cytokine genes in macrophages exposed to endotoxin (LPS). The MEK → ERK kinase pathway has been shown to be required for the activation of AP-1 and C/EBPβ, both of which are transcription factors involved in the transcription of cytokine genes (25Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (521) Google Scholar, 26Minden A. Lin A. Smeal T. Derijard B. Cobb M. Davis R. Karin M. Mol. Cell. Biol. 1994; 14: 6683-6688Crossref PubMed Scopus (440) Google Scholar, 27Frost J.A. Geppert T.D. Cobb M.H. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3844-3848Crossref PubMed Scopus (205) Google Scholar, 28Frost J.A. Alberts A.S. Sontag E. Guan K. Mumby M.C. Feramisco J.R. Mol. Cell. Biol. 1994; 14: 6244-6252Crossref PubMed Scopus (93) Google Scholar, 29DeSilva D.R. Feeser W.S. Tancula E.J. Scherle P.A. J. Exp. Med. 1996; 183: 2017-2023Crossref PubMed Scopus (113) Google Scholar). Another study has shown that sustained activation of the MEK → ERK pathway resulted in induction of IL-5, IL-6, IL-10, and IL-13 genes in T cells (30Chen D. Heath V. O'Garra A. Johnston J. McMahon M. J. Immunol. 1999; 163: 5796-5805PubMed Google Scholar). None of these studies, however, evaluated the effect of the MEK → ERK pathway on NF-κB-dependent transcription. Based on our previous data showing that the ERK kinase regulated IL-6 transcription (24Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell. Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar) and that the p38 MAP kinase was necessary for NF-κB-dependent gene expression (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), we analyzed the role of the MEK → ERK pathway in regulating NF-κB-dependent transcription. By utilizing a promoter construct driven only by NF-κB, we found that a constitutive active MEK1 expression vector inhibited NF-κB-dependent luciferase activity in LPS-stimulated THP-1 cells, and PD 98059, a specific inhibitor of MEK, abrogated this inhibition. NF-κB activation was not altered by the MEK → ERK pathway. In contrast, the constitutive active MEK1 expression vector inhibited the phosphorylation of a His-TBP fusion protein, which must be phosphorylated to interact with the p65 subunit and to bind to the TATA box. In addition, the constitutive active MEK → ERK pathway reduced the activity of the p38 MAP kinase, suggesting that the ERK and p38 MAP kinases have differential effects on NF-κB-dependent gene expression. The THP-1 cell line was obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with gentamicin and 10% fetal calf serum (Life Technologies, Inc.). For in vivo phosphorylation studies the cells were cultured in phosphate-free RPMI 1640 medium (Life Technologies, Inc.) with the same supplements, and for in vivo kinase assays the cells were cultured in RPMI 1640 medium supplemented with gentamicin and 0.5% fetal calf serum. NF-κB-dependent gene expression was evaluated utilizing a luciferase reporter plasmid (pNF-κB-luc) driven by four tandem copies of the κ enhancer (κB4) in a pUC vector (CLONTECH, Palo Alto, CA). The −600 TNF-CAT (a generous gift from Dr. Tom Maniatis, Harvard University, Cambridge, MA) and −546 IL-8-CAT (a generous gift from Dr. Naofumi Mukaida, Kanazawa University, Kanazawa, Japan) plasmids have been previously described (31Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar, 32Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Abstract Full Text PDF PubMed Google Scholar). The pCMV-HA-ERK2 (K/A), pCMV-MEK1 plasmids (generous gifts from Dr. Roger Davis, University of Massachusetts, Worcester, MA), pcDNA-His-p65, and pcDNA-His-TBP plasmids have been described previously (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 33Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar). The pcDNA-V5-p38 plasmid was generated by amplification of the human open reading frame of the p38 MAP kinase and inserting it into a pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA). The pTET-luc and pTET-Elk plasmids were obtained from CLONTECH. Transfections were performed utilizing the Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's recommendations. Twenty four h after transfection the cells were stimulated with Escherichia coli serotype 026:B6 LPS (Sigma) at a dose of 100 μg/ml. Luciferase activity, which was normalized to total protein, was measured after 6 h (Promega, Madison, WI), which was determined to be the time of maximal activity. Chloramphenicol acetyltransferase (CAT) assays, which were normalized to total protein, were performed after 24 h as described previously (34Hunninghake G.W. Monick M.M. Liu B. Stinski M.F. J. Virol. 1989; 63: 3026-3033Crossref PubMed Google Scholar). When used, the MEK kinase inhibitor, PD 98059 (Calbiochem), at 5 μm, was added 1 h prior to stimulation with LPS. A consensus NF-κB (5′-AGTTGAGGGGATTTTCCCAGGC-3′) oligonucleotide (Promega) was labeled with [γ-32P]ATP (NEN Life Science Products). Binding reactions were performed as described previously (35Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell. Mol. Biol. 1998; 18: 384-391Crossref PubMed Scopus (115) Google Scholar, 36Monick M.M. Carter A.B. Hunninghake G.W. J. Biol. Chem. 1999; 274: 18075-18080Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and the protein-DNA complexes were separated on a 5% polyacrylamide gel. Whole cell lysates were prepared as described previously (24Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell. Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar, 37Monick M.M. Carter A.B. Gudmundsson G. Mallampalli R. Powers L.S. Hunninghake G.W. J. Immunol. 1999; 162: 3005-3012PubMed Google Scholar). The p38 or ERK2 MAP kinases were immunoprecipitated from the lysates overnight at 4 °C with either a p38 or ERK2 polyclonal rabbit antibodies (Santa Cruz Biotechnology), respectively, bound to Gammabind with Sepharose (Amersham Pharmacia Biotech). In vitro kinase activity was assayed as described previously using ATF-2, c-Jun, or TFIID (TBP) (Santa Cruz Biotechnology) as a substrate (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 24Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell. Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar, 37Monick M.M. Carter A.B. Gudmundsson G. Mallampalli R. Powers L.S. Hunninghake G.W. J. Immunol. 1999; 162: 3005-3012PubMed Google Scholar). Co-transfection of the pTET-luc with the pTET-ELK plasmid was performed to determine kinase activity in vivo. After 24 h, the cells were stimulated with LPS, and luciferase activity, which was normalized to protein, was measured after 6 h, which was determined to be the time of maximal activity. For Western blot analysis, SDS-polyacrylamide gels were transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) as described previously (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). ERK2 and p38 MAP kinase rabbit polyclonal antibodies were used at 1:1000 dilution, and the p-ERK monoclonal antibody (Santa Cruz Biotechnology) was used at 1:500 dilution. The anti-Xpress monoclonal and the anti-V5 monoclonal antibodies (Invitrogen) recognize the sequences -Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys- and -Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr-, respectively, from the leader peptide in the His-p65, His-TBP, and the V5-p38 fusion proteins, and they were used at 1:1000 dilution. Immunoreactive proteins were developed using a chemiluminescent substrate (Amersham Pharmacia Biotech). THP-1 cells were co-transfected with an empty vector or the pCMV-MEK1 and either pcDNA-His-p65, pcDNA-His-TBP, or pcDNA-V5-p38 plasmids. After 24 h, the cells were labeled as described previously (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The His-p65, the His-TBP, or the V5-p38 fusion proteins were immunoprecipitated from whole cell lysates with the anti-Xpress or anti-V5 monoclonal antibody bound to Gammabind with Sepharose (Amersham Pharmacia Biotech) from 2 h to overnight at 4 °C. The samples were separated by SDS-PAGE, and phosphorylation was determined by autoradiography. All luciferase activity assays, which were normalized to total protein, are shown as means with the standard error. Statistical comparisons were performed using a paired, one-tailed t test, with the probability of p< 0.05 considered to be significant. Prior to determining the role of the ERK kinase in regulating NF-κB-dependent gene transcription, we assured ourselves that LPS activates the ERK kinase and that PD 98059 was working appropriately in THP-1 cells. To demonstrate that LPS activates the ERK kinase, cells were stimulated with LPS at various time points, and a Western analysis was performed for p-ERK. We found that p-ERK was maximal at 30 min, but there was increased activity as early as 5 min, and it gradually decreased to control levels at 90 min (Fig.1 A). Western blot analysis of ERK2 MAP kinase shows equal loading of the protein. In vitrokinase assays were performed using c-Jun as a substrate, and similar results were found (data not shown). Interestingly, other isoforms of ERK kinase have minimal activity in LPS-stimulated THP-1 cells when compared with the ERK2 kinase (data not shown). These data show that LPS activates the ERK2 kinase in THP-1 cells. The MEK inhibitor, PD 98059, has been shown previously to be an effective and relatively specific inhibitor of ERK-mediated signaling (38Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2601) Google Scholar, 39Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3262) Google Scholar, 40Zhang C. Baumgartner R.A. Yamada K. Beaven M.A. J. Biol. Chem. 1997; 272: 13397-13402Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In order to demonstrate that PD 98059 was working appropriately in THP-1 cells, we utilized both in vivo andin vitro kinase assays to quantify activation of either the ERK2 or the p38 MAP kinase. We co-transfected a luciferase reporter with the pTET-ELK plasmid and performed luciferase assays. LPS significantly increased luciferase activity in cells expressing pTET-ELK, and in the presence of PD 98059, this activity was reduced to control levels (Fig. 1 B). Similar results were obtained performing Western analysis for p-ERK and in ERK2 in vitrokinase assays using c-Jun as the substrate (data not shown). In contrast, LPS significantly increased p38 MAP kinase activity as measured by phosphorylation of ATF-2, and PD 98059 had no appreciable effect on kinase activity. Interestingly, PD 98059 slightly increased this activity above base line in cells not exposed to LPS (Fig.1 C). These studies confirm that PD 98059 is effective and relatively specific for ERK-mediated signaling. Furthermore, inhibition of the MEK → ERK pathway with PD 98059 slightly enhanced p38 MAP kinase-mediated signaling as quantified by kinase activity assays. To determine the role of the MEK → ERK pathway in regulating LPS-induced NF-κB-dependent transcription, we measured NF-κB-dependent promoter activity using the pNF-κB-luc reporter plasmid. We first the used thein vivo kinase assay to show that pCMV-MEK1 was constitutively active in THP-1 cells. Cells were co-transfected with the luciferase reporter plasmid, pTET-ELK, and either an empty vector or the constitutive active MEK1 expression vector. Both LPS and MEK1 increased luciferase activity greater than 4× control, and in cells expressing MEK1 and stimulated with LPS the luciferase activity was at a similar level (Fig. 2 A). An ERK2 in vitro kinase assay using c-Jun as the substrate revealed similar results (data not shown). These data confirm that pCMV-MEK1 constitutively activates ERK2 both in vivo andin vitro. By utilizing the constitutive active MEK1 expression vector, we next evaluated its role in regulating NF-κB-driven gene transcription. In these studies, we co-transfected the pNF-κB-luc with either an empty vector or the MEK1 expression vector. LPS significantly increased luciferase activity in cells expressing the empty vector, whereas in cells expressing the constitutive active MEK1 vector, luciferase activity was reduced to near control levels (Fig. 2 B). A similar experiment was performed with cells cultured in the presence or absence of PD 98059 after the transient co-transfection. PD 98059 augmented LPS-induced luciferase activity in cells expressing the empty vector, and in cells expressing the constitutive active MEK1 vector, PD 98059 abrogated the effect of MEK1 on NF-κB-dependent luciferase activity (Fig. 2 C). To evaluate this in a different manner, we co-transfected the pNF-κB-luc plasmid with either an empty vector or the dominant negative pCMV-HA-ERK2 (K/A) plasmid. The results from these experiments corroborated the results found with PD 98059. LPS increased luciferase activity in cells transfected with the empty vector, and this activity was augmented in cells expressing the dominant negative ERK2 MAP kinase (Fig. 2 D). Taken together, these results show that the MEK → ERK pathway negatively regulates NF-κB-dependent gene expression. In order to determine the role of the MEK → ERK kinase pathway in the regulation NF-κB-driven transcription, we next evaluated if either translocation or p65 phosphorylation was affected by MEK1 or ERK2 activity. THP-1 cells were cultured in the presence or absence of PD 98059 for 1 h and then stimulated with LPS, and nuclear protein was subsequently extracted. An electrophoretic mobility shift assay showed that LPS increased NF-κB translocation and DNA binding. Inhibition of the MEK → ERK pathway with PD 98059 had essentially no effect on this translocation and binding (Fig. 3 A). To evaluate if the constitutive active MEK1 altered p65 phosphorylation, cells were co-transfected with the pcDNA-His-p65 plasmid and either an empty vector or the constitutive active MEK1 expression vector. After 24 h, the cells were labeled with32Pi in phosphate-free medium and then stimulated with LPS. His-p65 was immunoprecipitated to assess phosphorylation. LPS increased His-p65 phosphorylation in cells expressing the empty vector, and the constitutive active MEK1 expression vector had no significant effect on this phosphorylation (Fig. 3 B). Western blot analysis of His-p65 shows equal loading of the immunoprecipitated proteins. As an aggregate, these studies indicate that the MEK → ERK pathway does not directly regulate the activation of NF-κB by controlling its translocation and DNA binding or the phosphorylation of the p65 subunit, and inhibition of this pathway with PD 98059 does not augment the translocation of NF-κB. Due to the fact that NF-κB-driven transcription requires activation of TBP and this activation is dependent on the p38 MAP kinase (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), we next evaluated if the negative regulation of NF-κB-dependent transcription by the MEK → ERK pathway was secondary to modulation of TBP activation. We co-transfected the pcDNA-His-TBP plasmid with either an empty vector or the constitutive active MEK1 expression vector. The cells were labeled with32Pi in phosphate-free medium and subsequently stimulated with LPS. His-TBP was immunoprecipitated from whole cell lysates to assess phosphorylation. LPS increased phosphorylation of His-TBP in cells expressing the empty vector. In contrast, His-TBP phosphorylation was completely obliterated in cells expressing MEK1 (Fig. 4). Western blot analysis of His-TBP shows equal loading of the immunoprecipitated proteins. This suggests that the MEK → ERK pathway negatively regulates NF-κB-dependent gene transcription, in part, by modulating the activation of TBP. To determine the physiological relevance of inhibiting NF-κB transcriptional activity by the constitutive active MEK1 expression vector, we utilized a CAT reporter gene driven by either a TNF-α or an IL-8 promoter, both of which have NF-κB-binding sites. THP-1 cells were co-transfected with an empty vector or pCMV-MEK1 and either −546 IL-8-CAT or −600 TNF-CAT. LPS significantly increased CAT activity in cells expressing the empty vector. In contrast, cells expressing the constitutive active MEK1 had about a 70% reduction, as measured by densitometry, in IL-8-CAT activity (Fig.5, A and B). In cells transfected with the TNF-CAT, LPS induced a significant increase in CAT activity, and this activity was reduced by about 40% in cells expressing the constitutive active MEK1 (Fig. 5 C). These data suggest the MEK → ERK pathway negatively regulates endogenous gene expression that requires NF-κB transcriptional activity by modulating TBP activation. Furthermore, the data suggest that cytokine gene expression is clearly dependent of NF-κB even though the MEK → ERK pathway is necessary for the activation of AP-1 and NF-IL-6, both of which have binding sites within the IL-8 and TNF promoters. Due to the fact that the p38 MAP kinase is a positive regulator (21Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) and the constitutive active MEK1 is a negative regulator of NF-κB-dependent transcription by affecting activation of TBP, we next evaluated if a constitutive active MEK → ERK pathway inhibited p38 MAP kinase activity. To determine if the constitutive active MEK1 expression vector inhibited the p38 MAP kinase, we first performed in vitro p38 MAP kinase assays. THP-1 cells were transfected with either an empty vector or the constitutive active MEK1 expression vector. The native p38 MAP kinase was immunoprecipitated from whole cell lysates, and kinase activity was determined using TFIID (TBP) as the substrate. TFIID (TBP) was phosphorylated in vitro by LPS-stimulated p38 MAP kinase obtained from THP-1 cells transfected with the empty vector, and this activity was significantly reduced in cells expressing MEK1 (Fig.6 A). Western blot analysis for p38 MAP kinase confirmed equal loading of the immunoprecipitated proteins. The dual phosphorylation of Thr-180 and Tyr-182 is required for p38 MAP kinase activation (41Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar), so we finally determined if the constitutive active MEK1 expression vector inhibited the phosphorylation of a p38 MAP kinase fusion protein. In these experiments, we co-transfected the pcDNA-V5-p38 plasmid with either an empty vector or the MEK1 expression vector. The cells were labeled with32Pi in phosphate-free medium prior to stimulation with LPS. The V5-p38 fusion protein was immunoprecipitated to determine phosphorylation. LPS induced the phosphorylation of V5-p38, and this phosphorylation was reduced in cells expressing MEK1 (Fig. 6 B). Western blot analysis for the V5-p38 fusion protein confirmed equal loading of the immunoprecipitated proteins. As an aggregate, these studies show that the p38 MAP kinase phosphorylates TFIID (TBP) and a constitutive active MEK → ERK pathway inhibits p38 MAP kinase activity both in vitro and in vivo by reducing its phosphorylation. These studies also provide novel evidence of a mechanism by which the MEK → ERK pathway, at least in part, negatively regulates NF-κB-dependent gene expression. NF-κB-dependent gene expression can be regulated at multiple levels after cell stimulation. The early regulation events occur in the cytoplasm where NF-κB is held by the inhibitor protein, IκB. Previous studies have shown that MEKK1, an upstream activator of both the JNK and p38 MAP kinase pathways, phosphorylates both IKKα and IKKβ, and it acts in conjunction with the NF-κB-inducing kinase to synergistically activate the IKK complex (42Nemoto S. DiDonato J.A. Lin A. Mol. Cell. 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More specifically, activation of the p38 and JNK MAP kinases and simultaneous inhibition of the ERK MAP kinase was found to be critical for apoptosis in PC-12 pheochromocytoma cells stimulated with nerve growth factor (51Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5074) Google Scholar). In another study, the p38 MAP kinase was shown to enhance accumulation of IL-12 mRNA, whereas the ERK MAP kinase inhibited IL-12 transcription in murine macrophages stimulated with LPS (52Feng G.J. Goodridge H.S. Harnett M.M. Wei X.Q. Nikolaev A.V. Higson A.P. Liew F.Y. J. Immunol. 1999; 163: 6403-6412PubMed Google Scholar). In addition to having opposing effects on gene expression, these kinases have also been shown to negatively regulate one another. For example, activation of the p38 MAP kinase with anisomycin in mast cells inhibited the activation of the ERK2 MAP kinase (40Zhang C. Baumgartner R.A. Yamada K. Beaven M.A. J. Biol. 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Although we did not determine the mechanism by which the constitutive active MEK1 expression vector inhibited p38 MAP kinase activity, one plausible mechanism would be through induction of MKP-1 or other phosphatases. The ERK MAP kinase has been shown to activate MKP-1 by phosphorylation on two carboxyl-terminal serines both in vivo and in vitro (54Brondello J.-M. Brunet A. Pouyssegur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 55Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (369) Google Scholar). Multiple studies have shown that MKP-1 also has much more specificity for the p38 MAP kinase than the other MAP kinases (56Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (576) Google Scholar, 57Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 58Charles C.H. Sun H. Lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. 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This may explain the mechanism by which both PD 98059 and the dominant negative ERK2 expression vector augment NF-κB-dependent gene expression. Our data clearly show that the constitutive activation of the MEK → ERK pathway negatively regulates NF-κB-dependent transcription through modulation of TBP activation. In addition, this is the first study showing that a constitutive active MEK → ERK pathway inhibits p38 MAP kinase activity and implicates the dynamic balance between ERK- and p38-mediated signaling required for NF-κB-dependent gene expression. It may be of interest in further studies to determine if the constitutive active MEK1 expression vector induces constitutive activation of a phosphatase. We thank the University of Iowa DNA Facility for sequencing the plasmids used in this study; Drs. Maniatis, Mukaida, and Davis for providing their respective plasmids; and P. Sankaman for outstanding technical assistance.

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