The MEK Pathway Is Required for Stimulation of p21WAF1/CIP1 by Transforming Growth Factor-β

Abstract

Transforming growth factor-β (TGF-β)can induce the cyclin-dependent kinase inhibitors p21 and p15 in a variety of cell types. We have shown previously that Smad3 is required for the growth inhibitory activity of TGF-β, whereas overexpression of Smads is not sufficient to activate the expression of p21 in HaCaT cells. These data suggest that an additional signaling pathway may be involved in stimulating p21 in HaCaT cells. Given the recent finding that the mitogen-activated protein kinase (MAPK) pathway can cause p21 induction and arrest cells, we examined the involvement of this pathway for p21 and p15 induction by TGF-β. We found that TGF-β can regulate the MAPK pathway, leading to the increased transactivation ability of transcription factor Elk. Constitutively active components in the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway significantly decrease p21 induction by TGF-β. Both constitutively active MEK and inhibitors for MEK have no effect on Smad activity, including DNA binding, localization, and interaction with coactivator p300/CBP. These findings suggest that the MAPK pathway may be an independent pathway that is involved in p21 and p15 induction by TGF-β. Transforming growth factor-β (TGF-β)can induce the cyclin-dependent kinase inhibitors p21 and p15 in a variety of cell types. We have shown previously that Smad3 is required for the growth inhibitory activity of TGF-β, whereas overexpression of Smads is not sufficient to activate the expression of p21 in HaCaT cells. These data suggest that an additional signaling pathway may be involved in stimulating p21 in HaCaT cells. Given the recent finding that the mitogen-activated protein kinase (MAPK) pathway can cause p21 induction and arrest cells, we examined the involvement of this pathway for p21 and p15 induction by TGF-β. We found that TGF-β can regulate the MAPK pathway, leading to the increased transactivation ability of transcription factor Elk. Constitutively active components in the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway significantly decrease p21 induction by TGF-β. Both constitutively active MEK and inhibitors for MEK have no effect on Smad activity, including DNA binding, localization, and interaction with coactivator p300/CBP. These findings suggest that the MAPK pathway may be an independent pathway that is involved in p21 and p15 induction by TGF-β. transforming growth factor-β mitogen-activated protein kinase glutathione S-transferase polyacrylamide gel electrophoresis Transforming growth factor-β is a cytokine that causes growth arrest in late G1 of the cell cycle (reviewed in Refs. 1Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: 105-150Crossref PubMed Scopus (508) Google Scholarand 2Hu P.P.-C. Datto M.B. Wang X.-F. Endocr. Rev. 1998; 19: 349-363Crossref PubMed Google Scholar). In the human keratinocyte cell line HaCaT, growth inhibition is thought to depend on the ability of TGF-β1 to increase synthesis of the cyclin-dependent kinase inhibitors p21 and p15. Increased p21 leads to an increase in its association with cyclin D-CDK4 and cyclin E-CDK2 and a decrease in cyclin-CDK complex activity. Increased p15 expression leads to an increase in association and sequestration of CDK4 and CDK6 from the regulatory cyclin partners. On the cell membrane, TGF-β ligand binds to the type II receptor, which recruits the type I receptor into the complex. The type I receptor is phosphorylated by the type II receptor kinase and, when activated, phosphorylates Smad2 and Smad3, allowing them to heteromerize with Smad4 and translocate into the nucleus. In the nucleus, Smad3 can bind to DNA with the consensus sequence GTCTAGAC that is found in the promoters of many TGF-β-regulated genes (3Zawel L. 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Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (230) Google Scholar), as well as to other proteins such as the transcriptional coactivators p300 and CBP (9Nishihara A. Hanai J.I. Okamoto N. Yanagisawa J. Kato S. Miyazono K. Kawabata M. Genes Cells. 1998; 3: 613-623Crossref PubMed Scopus (130) Google Scholar, 10Shen X. Hu P.P.-C. Liberati N.T. Datto M.B. Frederick J.P. Wang X.-F. Mol. Biol. Cell. 1998; 9: 3309-3319Crossref PubMed Scopus (185) Google Scholar, 11Janknecht R. Wells N.J. Hunter T. Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (437) Google Scholar, 12Feng X.-H. Zhang Y. Wu R.-Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (451) Google Scholar) in a phosphorylation-dependent manner. Smad3 is also required for TGF-β-mediated growth inhibition because murine embryonic fibroblasts deficient in Smad3 have lost their ability to become growth inhibited by TGF-β (13Datto M.B. Frederick J.P. Pan L. Borton A.J. Zhuang Y. Wang X.F. Mol. Cell. Biol. 1999; 19: 2495-2504Crossref PubMed Google Scholar). Although Smads are required for TGF-β-mediated growth arrest, the mechanism underlying their action is still unknown. In HaCaT cells, TGF-β induction of the cyclin-dependent kinase inhibitors p21 and p15 correlates with both Smad nuclear translocation as well as growth arrest, but when Smad3 and Smad4 are overexpressed, they are unable to activate p21 and p15 transcription. In contrast, overexpression of Smad3 and Smad4 is sufficient to activate transcription of plasminogen activator inhibitor (14Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar), which contains Smad-binding elements in its promoter region (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar, 15Stroschein S.L. Wang W. Luo K. J. Biol. Chem. 1999; 274: 9431-9441Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This suggests that even though Smads may be required for growth inhibition by TGF-β, they are not sufficient to drive the transcription of p21 and p15, indicating that other pathways may also be needed for the transcriptional induction of p21 and p15 upon TGF-β treatment. Contrary to their role as the mediators of many mitogens, the MAPKs have been recently implicated in cellular growth arrest and senescence. Overexpression of Raf leads to growth arrest by impinging directly on the cell cycle through increasing p21 expression, leading to its association and concomitant inhibition of CDKs (16Lloyd A.C. Obermuller F. Staddon S. Barth C.F. McMahon M. Land H. Genes Dev. 1997; 11: 663-677Crossref PubMed Scopus (206) Google Scholar, 17Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (577) Google Scholar). In addition, altering the cellular ratio of existing Ras and Rho proteins can also lead to stimulation of p21 (18Olson M.F. Paterson H.F. Marshall C.J. Nature. 1998; 394: 295-299Crossref PubMed Scopus (410) Google Scholar). TGF-β can also increase GTP-bound Ras, and activate MAPK pathway in other cell types (19Mulder K.M. J. Biol. Chem. 1992; 267: 5029-5031Abstract Full Text PDF PubMed Google Scholar). Because activation of the Ras-Raf-MEK-MAPK pathway can induce transcription of p21, in this study we asked whether this pathway was involved in the ability of TGF-β to stimulate p21 and p15 expression. Here, we show that overexpression of constitutively active forms of different components in the MAPK cascade can substitute for the ability of TGF-β to stimulate p15 and p21 reporter activity. Stable lines overexpressing a constitutively active form of MEK, MEK Q56P (20Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (771) Google Scholar), also have increased expression of p21. Furthermore, inhibitors of this pathway prevent TGF-β induction of p21 and reduce TGF-β-mediated growth arrest in parental HaCaT cells. In contrast, TGF-β activation of Smad signaling is not affected by these inhibitors. Taken together, these data suggest that the MAPK pathway is involved in p21 up-regulation by TGF-β. The human p21 and p15 minimal promoter constructs, p21P93S and p15P113S, have been described previously (21Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). GAL4-Sp1 and GAL4-Sp3 full-length, GAL4-Sp1, and Sp3 B domain constructs have been were described previously (22Li J.M. Datto M.B. Shen X. Hu P.P. Yu Y. Wang X.F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar). GAL4-Elk, Ras 17A, Raf-N4, Raf-CAAX, MEK S218/222D, Rac 17N, cdc42 17N, MKK4/SEK-1, and SEK-AL constructs were the generous gift of Dr. Channing Der. The MEK Q56P pBABE-puro construct was the generous gift of Dr. Scott Lowe. HaCaT cells were the gift of Dr. Baukamp and Dr. Fusenig. HaCaT were maintained in 10% fetal bovine serum (Life Technologies, Inc) in α-minimum essential medium (Life Technologies, Inc.). For luciferase assays, cells were lysed, and luciferase activity in the lysates were assayed by integrating total light emission over 30 s using a Berthold luminometer. The luciferase activities were normalized based on the amount of expressed β-galactosidase activity expressed from co-transfected pCMV-β-galactosidase. Cells were lysed in universal lysis buffer (ULB) as described previously (10Shen X. Hu P.P.-C. Liberati N.T. Datto M.B. Frederick J.P. Wang X.-F. Mol. Biol. Cell. 1998; 9: 3309-3319Crossref PubMed Scopus (185) Google Scholar). Equal protein amounts of each sample were resolved on a 12% acrylamide-0.5% bisacrylamide sodium dodecyl sulfate gel. Proteins were transferred to Immobilon P transfer membrane (Millipore Corp.). Blocking and antibody incubations were performed in 5% dried milk and 0.1% Tween 20 in phosphate-buffered saline for 1 h at room temperature. Antibodies used were: anti-p21 (Santa Cruz Biotechnology, C-19), p15 (Santa Cruz Biotechnology, C-20), anti-Smad-3 (Santa Cruz Biotechnology, H-2), and horseradish peroxidase-conjugated goat-anti-rabbit (Bio-Rad). Western blots were developed with ECL reagent (Amersham Pharmacia Biotech). Complementary oligonucleotides corresponding to two copies of the Smad3-binding consensus element (GTCTAGAC) were synthesized. Electrophoretic mobility shift assays and nuclear extracts preparation were performed as described previously (8Wong C. Rougier-Chapman E. Frederick J. Datto M. Liberati N. Li J.-M. Wang X.-F. Mol. Cell. Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (230) Google Scholar). The MEK Q56P DNA construct inserted into the retroviral vector pBABE puro was a generous gift from Scott Lowe. Amphotrophic pBabe-puro-MEK Q56P retrovirus was then created using standard techniques (23Counter C.M. Hahn W.C. Wei W. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Crossref PubMed Scopus (564) Google Scholar). A control retrovirus was also generated. HaCaT cells were infected with the retroviruses and grown 10% fetal bovine serum α-minimum essential medium supplemented with 0.6 μg/ml puromycin for (pBabe-puro-MEK Q56P) or 300 μg/ml hygromycin (control) for several days. Cells were plated on coverslips and grown overnight. The next day, cells were treated with TGF-β for 30 min, fixed in paraformaldehyde for 10 min at room temperature, and permeabilized in 0.5% Triton X-100 for 5 min. Slips were then incubated in primary (anti-Smad-3, Santa Cruz Biotechnology, H-2) and secondary antibody for 1 h at 37 °C with frequent washes. Slips were then mounted and photographs taken on a Zeiss confocal microscope. Approximately 200 μg of proteins from cells lysed in ULB were incubated with protein A (Amersham Pharmacia Biotech) and α-Smad3 (Santa Cruz Biotechnology) for immunoprecipitation experiments or glutathione-Sepharose bound GST-p300C (20 μg) for GST pull-down experiments. Binding reactions were for 2 h at 4 °C with continual rotation. The beads were collected and washed three times (3 min/wash) with ULB. Bound proteins were eluted by boiling in 1× Laemmli sample buffer, separated by SDS-PAGE, and subjected to immunoblot analysis. Activation of the MAPK pathway leads to phosphorylation of transcription factor such as and and their transactivation (reviewed in Refs. R. Cell. 1995; Full Text PDF PubMed Scopus Google and R.J. Mol. Dev. 1995; PubMed Scopus Google Scholar). to be a for in and its phosphorylation with MAPK whether MAPK pathway is upon TGF-β a to and a reporter in its promoter were into HaCaT shown in TGF-β of cells leads to increased luciferase expression from the This suggests that TGF-β is to regulate and activate the MAPK pathway in HaCaT cells. Although overexpression of Smad3 and Smad4 was not sufficient to induce p21 reporter activity, the recent that activation of the Ras pathway stimulate p21 expression to this in HaCaT cells, for TGF-β whether the MAPK pathway was sufficient to induce p21 reporter activity, we a constitutively form of MEK in HaCaT cells and treated the cells with TGF-β. shown in 2 overexpression of the not p21 and p15 reporter activity in the of TGF-β but also for the TGF-β that TGF-β signaling may with this pathway to stimulate p21 transcription. Because the element in the p21 promoter been to a element (21Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) and GAL4-Sp1 but not GAL4-Sp3 (22Li J.M. Datto M.B. Shen X. Hu P.P. Yu Y. Wang X.F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar) can transcription when using the we whether MEK stimulate GAL4-Sp1 or transcription. shown in 2 GAL4-Sp1 but not GAL4-Sp3 transcription activation with expression of the constitutively active MEK This the effect of TGF-β. whether overexpression of other of the Ras-Raf-MEK-MAPK pathway were also to induce p15 and p21 promoter we performed experiments with a constitutively form of Raf and Ras For both p21 and the constitutively active form of Raf transcription also transcription from p21 the effect was not as as that of not This in the ability of Ras and to activate the p21 promoter may be the of a in the expression of proteins or their activity. In contrast, overexpression of and dominant not or p21 reporter activity whether overexpression of MEK increase expression of p21, we used a retroviral construct a MEK Q56P that is constitutively active (20Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (771) Google Scholar). Stable of infected human fibroblasts with this retrovirus with puromycin of p21 (20Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (771) Google Scholar). were in of HaCaT cells with an increase in p21 protein the p21 are in these cells, they and to as as parental HaCaT cells. these cells are and to be an increase in not suggest that TGF-β and other such as growth factor and growth factor can induce and nuclear through the MAPKs C.J. P. Roberts A.B. Lechleider R.J. Genes Dev. 1998; 12: PubMed Scopus Google Scholar). suggest that activation of the MAPKs can lead to a Smad phosphorylation that its nuclear M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar). MEK HaCaT cells, we were to whether constitutively active MEK Smad signaling and to whether the MAPK pathway of Smad signaling or and Smad We found that HaCaT cells infected with MEK Q56P in to Smad activity. TGF-β an complex is still in the mobility shift with the Smad-binding element as a In addition, the overexpression of MEK Q56P in cells not the ability of Smads to translocate into the upon TGF-β whether TGF-β growth inhibitory effect on cells, the amount of a was h TGF-β treatment. Smad signaling not affected by MEK Q56P is not that TGF-β can still the of cells whether the Ras-Raf-MEK-MAPK cascade was required for the ability of TGF-β to induce p21, p15, as well as dominant negative Ras or Raf were in HaCaT cells with the p21 minimal promoter reporter the p15 minimal promoter reporter or with the minimal reporter and treated with TGF-β for shown in 4 the ability of TGF-β to stimulate p21 and p15 promoters was by of the dominant negative dominant negative a effect on the p15 but to p21 promoter the effect is not as as that of the Ras dominant negative 4 this in the ability of Ras and dominant negative to the TGF-β-mediated induction of promoters may be the of a in the expression of proteins or their activity. these suggest that both Ras and Raf are required for TGF-β to stimulate promoter activities of p15 and p21. the experiments were performed with a dominant negative as well as a Rac dominant in both the ability of TGF-β to induce the p21 reporter was not affected 4 This is to a previously study a dominant negative Ras can the ability of to induce and p21 in cells J. A. K.M. 1998; 17: PubMed Scopus Google Scholar). we asked whether MEK was required for TGF-β to stimulate the expression of p21 and p15. A MEK was recently developed and found to be the MEK at MEK and activity, the activity of other and The of this to the in of MEK activity in TGF-β stimulation of p21 and p15 expression. Cells were treated with at of 10 or for 30 min with TGF-β for shown in 4 the ability of TGF-β to induce p21 and p15 protein expression was in the of the In contrast, and of p21 and p15 were The ability of Smad3 to bind to Smad4 upon TGF-β 4 and the ability of Smad3 to bind to GST-p300C 4 were not affected by with that a of TGF-β type I receptor kinase activity not at the of for the inhibitor In addition, not with of not that this inhibitor as a the of the of TGF-β to induce p21 and p15 expression, we the ability of TGF-β to cause growth inhibition in the of the inhibitor using cells were treated with TGF-β in the of increasing of for h and with for the 2 shown in 4 the ability of TGF-β to growth is with increasing of In addition, the cells treated with not the with TGF-β not and of suggest that Ras activation may be a of the TGF-β signaling pathway required for p21 The Ras activation and the TGF-β receptor unknown. Because mechanism for the activation of Ras by many the activation of we this by using an inhibitor of kinase activity S. S. R.J. Y. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). shown in the ability of TGF-β to increase p21 protein expression is significantly upon of HaCaT cells with 1 that a kinase may be involved in the TGF-β A is the signaling cascade to induce p21 and cause growth In cells, such as TGF-β, growth growth of a the and the I inhibitor (21Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, H. T. K. J. Cell. 1999; PubMed Scopus Google Scholar, N. D. Datto M.B. van A. Wang X.F. 1999; PubMed Scopus Google Scholar, Y. M.B. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, T. L. A. C. Datto M.B. Wang X.F. M. Res. 1999; PubMed Scopus Google Scholar, J. Y. Mol. Cell. Biol. 1998; PubMed Google Scholar, J. D. A.B. 1995; 267: PubMed Scopus Google Scholar, C. E. R. J. Proc. Natl. Acad. Sci. U. S. A. 1995; Scholar) have been shown to induce p21 expression as well as growth In addition, of these to through and elements in the p21 Activation of the MAPK pathway be a pathway these may to stimulate p21 expression. in an a pathway is activated, in the phosphorylation of the transcription factor a increase in transcription of the of a cyclin-dependent kinase and cell cycle arrest Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). phosphorylation of by the MAPKs may be mechanism by which transcription of p21 is increased in these cells. TGF-β can activate different MAPK to stimulate transcriptional In a recent by EMBO J. 1999; PubMed Google Scholar), TGF-β was shown to stimulate activity to increase synthesis in a manner. in activity have been A. S. E. R. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), as have in K.M. Res. 1997; Google Scholar, K.M. 1997; PubMed Scopus Google Scholar). We have taken of the for and and performed Western blots to which MAPK is by TGF-β in Although transcription is increased upon TGF-β in HaCaT cells these three MAPK not to be significantly by TGF-β not This suggests that other proteins may be by TGF-β. Although a MAPK kinase kinase to TGF-β, is such K. K. H. K. N. E. E. K. 1995; 270: PubMed Scopus Google Scholar), expression of a dominant negative of kinase not the ability of TGF-β to stimulate p21 expression not is to on TGF-β on the MAPK can activate MAPK signaling through different In kinase receptor upon of ligand such as growth the receptor protein kinase is and recruits such as and in with Ras and which in Ras In the become of from which can then activate a cascade Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). The ability of kinase to prevent TGF-β induction of p21 suggests that a kinase activity is required for its Although on the TGF-β type II receptor may be phosphorylated and (reviewed in C.-H. Miyazono K. ten Dijke P. Nature. 1997; PubMed Scopus Google Scholar), to be whether these to TGF-β signaling Smad proteins a role in TGF-β signaling pathway, and we show that MAPK pathway is also essential for p21 induction by TGF-β. Smads to the MAPK pathway and growth arrest activity for TGF-β is still experiments suggest that Smads are required for TGF-β-mediated growth arrest but on the other overexpression of Smads may not be sufficient for the p21 data that the MAPK pathway not of Smads because cells overexpressing constitutively MEK Q56P not show an effect on Smad or DNA the MAPK pathway be a pathway from Smad a role as a of Smad the is the at in to Smads is needed to activate the In cells, this other may be constitutively activated, as is the in cells Smads can stimulate p21 promoter activity A. D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). Because in HaCaT cells activation of the MAPK pathway but not is sufficient to stimulate p21 expression and in cells overexpression of Smads can also stimulate p21, is that the MAPK pathway of is that Smad3 can also as a required in to its role in the that the association the TGF-β and In this a recent that Smad3 may with other proteins such as to TGF-β signaling T. L. J.L. Cell. 1998; 95: Full Text Full Text PDF PubMed Scopus Google Scholar). We for We are to Dr. J. for the Dr. C. for the GAL4-Elk, Ras 17A, Raf-N4, Raf-CAAX, Rac 17N, cdc42 17N, MKK4/SEK-1, and SEK-AL constructs, and Dr. Scott Lowe for the MEK Q56P pBABE-puro In addition, we R. P. K. S. and A. M. for and we of the Wang for and Yu for