Cyclic AMP Promotes cAMP-responsive Element-binding Protein-dependent Induction of Cellular Inhibitor of Apoptosis Protein-2 and Suppresses Apoptosis of Colon Cancer Cells through ERK1/2 and p38 MAPK

Abstract

We recently reported that cAMP suppresses apoptosis in colon cancer cells and induces cellular inhibitor of apoptosis protein-2 (c-IAP2) via a cAMP-responsive element (CRE), suggesting a mechanism for chemoprevention of colon cancer by non-steroidal anti-inflammatory drugs. In this study, we used T84 human colon cancer cells to define the pathway by which increases in cAMP induce c-IAP2 expression. Treatment with several different cAMP agonists stimulated phosphorylation of CRE-binding protein (CREB) and activated expression of c-IAP2 in a CREB-dependent manner. Studies with pharmacological inhibitors revealed that cAMP-dependent phosphorylation of CREB required activation of ERK1/2 and p38 MAPK but was largely independent of protein kinase A. Immunoblots and transcriptional reporter assays using specific inhibitors, as well as expression of constitutively active forms of MEK1 and MKK3, showed that c-IAP2 induction by cAMP is regulated predominantly through ERK1/2 and p38 MAPK and suggested involvement of p90 ribosomal protein S6 kinase and mitogen and stress response kinase-1 as well. Consistent with those results, we found that cAMP-dependent suppression of apoptosis was blocked by treatment with inhibitors of ERK1/2 and p38 MAPK. We conclude that cAMP can induce c-IAP2 expression in colon cancer cells through CREB phosphorylation and CRE-dependent transcription in a manner that involves activation of ERK1/2 and p38 MAPK. These results emphasize that activation of kinases other than protein kinase A can mediate the actions of agents that increase cAMP, particularly in the regulation of CREB-dependent events. We recently reported that cAMP suppresses apoptosis in colon cancer cells and induces cellular inhibitor of apoptosis protein-2 (c-IAP2) via a cAMP-responsive element (CRE), suggesting a mechanism for chemoprevention of colon cancer by non-steroidal anti-inflammatory drugs. In this study, we used T84 human colon cancer cells to define the pathway by which increases in cAMP induce c-IAP2 expression. Treatment with several different cAMP agonists stimulated phosphorylation of CRE-binding protein (CREB) and activated expression of c-IAP2 in a CREB-dependent manner. Studies with pharmacological inhibitors revealed that cAMP-dependent phosphorylation of CREB required activation of ERK1/2 and p38 MAPK but was largely independent of protein kinase A. Immunoblots and transcriptional reporter assays using specific inhibitors, as well as expression of constitutively active forms of MEK1 and MKK3, showed that c-IAP2 induction by cAMP is regulated predominantly through ERK1/2 and p38 MAPK and suggested involvement of p90 ribosomal protein S6 kinase and mitogen and stress response kinase-1 as well. Consistent with those results, we found that cAMP-dependent suppression of apoptosis was blocked by treatment with inhibitors of ERK1/2 and p38 MAPK. We conclude that cAMP can induce c-IAP2 expression in colon cancer cells through CREB phosphorylation and CRE-dependent transcription in a manner that involves activation of ERK1/2 and p38 MAPK. These results emphasize that activation of kinases other than protein kinase A can mediate the actions of agents that increase cAMP, particularly in the regulation of CREB-dependent events. Suppression of normal apoptotic pathways contributes to tumor progression and confers resistance to cytotoxic anticancer drugs and radiation (1Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar). The second messenger cAMP has antiapoptotic actions (2Boucher M.J. Duchesne C. Laine J. Morisset J. Rivard N. Biochem. Biophys. Res. Commun. 2001; 285: 207-216Crossref PubMed Scopus (40) Google Scholar, 3Garcia-Bermejo L. Perez C. Vilaboa N.E. de Blas E. Aller P. J. Cell Sci. 1998; 111: 637-644Crossref PubMed Google Scholar, 4Baumann C.A. Badamchian M. Goldstein A.L. Mech. Ageing Dev. 1997; 94: 85-101Crossref PubMed Scopus (46) Google Scholar), and its primary effector enzyme, PKA, 1The abbreviations used are: PKA, protein kinase A; IAP, inhibitor of apoptosis proteins; c-IAP, cellular IAP; CRE, cAMP-responsive element; CREB, CRE-binding protein; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; RSK, ribosomal protein S6 kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKK, MAPK kinase; MSK, mitogen and stress response kinase; PGE2, prostaglandin E2; BIR, baculoviral IAP repeat; TNF, tumor necrosis factor; mAb, monoclonal antibody; CTX, cholera toxin; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; Fsk, forskolin; (Rp)-cAMPS, adenosine 3′,5′-cyclic monophosphothioate, Rp isomer; 8-CPT-2 ′-O-Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; WT, wild type; CHO, Chinese hamster ovary; ATF-2, activating transcription factor-2.1The abbreviations used are: PKA, protein kinase A; IAP, inhibitor of apoptosis proteins; c-IAP, cellular IAP; CRE, cAMP-responsive element; CREB, CRE-binding protein; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; RSK, ribosomal protein S6 kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKK, MAPK kinase; MSK, mitogen and stress response kinase; PGE2, prostaglandin E2; BIR, baculoviral IAP repeat; TNF, tumor necrosis factor; mAb, monoclonal antibody; CTX, cholera toxin; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; Fsk, forskolin; (Rp)-cAMPS, adenosine 3′,5′-cyclic monophosphothioate, Rp isomer; 8-CPT-2 ′-O-Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; WT, wild type; CHO, Chinese hamster ovary; ATF-2, activating transcription factor-2. is a target for cancer therapy (5Tortora G. Ciardiello F. Ann. N. Y. Acad. Sci. 2002; 968: 139-147Crossref PubMed Scopus (90) Google Scholar, 6Tortora G. Ciardiello F. Clin. Cancer Res. 2002; 8: 303-304PubMed Google Scholar, 7Cho Y.S. Kim M.K. Tan L. Srivastava R. Agrawal S. Cho-Chung Y.S. Clin. Cancer Res. 2002; 8: 607-614PubMed Google Scholar). Prostaglandin E2 (PGE2), which is formed from arachidonic acid by cyclooxygenases, binds to Gs-coupled receptors and increases intracellular cAMP concentration (8Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). Cyclooxygenase levels are high in human colon cancers (9Rigas B. Goldman I.S. Levine L. J. Lab. Clin. Med. 1993; 122: 518-523PubMed Google Scholar), and cyclooxygenase inhibition prevents cell proliferation and promotes apoptosis (10Chan T.A. Lancet Oncol. 2002; 3: 166-174Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 11Leahy K.M. Ornberg R.L. Wang Y. Zweifel B.S. Koki A.T. Masferrer J.L. Cancer Res. 2002; 62: 625-631PubMed Google Scholar). Recently we reported that increases in cAMP levels promoted by PGE2 or other agents that raise cAMP inhibit apoptosis in colon cancer cells through the induction of c-IAP2, therefore suggesting a novel mechanism of cancer chemoprevention by non-steroidal anti-inflammatory drugs (12Nishihara H. Kizaka-Kondoh S. Insel P.A. Eckmann L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8921-8926Crossref PubMed Scopus (118) Google Scholar). Inhibitors of apoptosis proteins (IAPs) are characterized by a domain of about 70 amino acids, termed the baculoviral IAP repeat (BIR), based on the original discovery of these apoptosis suppressors in the baculoviral genome (13Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar, 14Birnbaum M.J. Clem R.J. Miller L.K. J. Virol. 1994; 68: 2521-2528Crossref PubMed Google Scholar). IAP family proteins have potentially important roles in the regulation of apoptosis and tumorigenesis (15Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2255) Google Scholar, 16Tamm I. Kornblau S.M. Segall H. Krajewski S. Welsh K. Kitada S. Scudiero D.A. Tudor G. Qui Y.H. Monks A. Andreeff M. Reed J.C. Clin. Cancer Res. 2000; 6: 1796-1803PubMed Google Scholar). The expression of survivin, one of the IAP family proteins, is significantly increased in several human cancers (17Hoffman W.H. Biade S. Zilfou J.T. Chen J. Murphy M. J. Biol. Chem. 2002; 277: 3247-3257Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Although eight human IAPs have been identified (18Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1563) Google Scholar), c-IAP1/BIRC2 and c-IAP2/BIRC3 are the only IAPs that appear to be part of a signaling complex recruited to the cytoplasmic domain of the type 2 TNF receptor (19Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1129) Google Scholar). In addition, c-IAP2 was also suggested to be a causative gene of mucosa-associated lymphoid tissue lymphoma and to have a role in carcinogenesis and tumor progression (20Dierlamm J. Baens M. Wlodarska I. Stefanova-Ouzounova M. Hernandez J.M. Hossfeld D.K. De Wolf-Peeters C. Hagemeijer A. Van den Berghe H. Marynen P. Blood. 1999; 93: 3601-3609Crossref PubMed Google Scholar). The active forms of caspase-3 and -7 are directly inhibited by c-IAP2 (19Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1129) Google Scholar), which can also prevent the proteolytic processing of procaspase-3, -6, and -7 by blocking the cytochrome c-induced activation of procaspase-9 (21Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1232) Google Scholar). Expression of c-IAP2 is regulated through multiple regulatory elements in its promoter region. A NF-κB binding site is essential for induction by TNFα in Jurkat T cells (22Chu Z.L. McKinsey T.A. Liu L. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (819) Google Scholar) and radiation in human embryonic kidney 293 cells (23Ueda T. Akiyama N. Sai H. Oya N. Noda M. Hiraoka M. Kizaka-Kondoh S. FEBS Lett. 2001; 491: 40-44Crossref PubMed Scopus (30) Google Scholar). Dexamethasone induces c-IAP2 expression through a putative glucocorticoid response element in A549 human lung cancer cells (24Webster J.C. Huber R.M. Hanson R.L. Collier P.M. Haws T.F. Mills J.K. Burn T.C. Allegretto E.A. Endocrinology. 2002; 143: 3866-3874Crossref PubMed Scopus (81) Google Scholar), while a cAMP-responsive element (CRE) has an essential role in induction of c-IAP2 expression by cAMP in T84 colon cancer cells (12Nishihara H. Kizaka-Kondoh S. Insel P.A. Eckmann L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8921-8926Crossref PubMed Scopus (118) Google Scholar). In this study, we analyzed the signal transduction pathways that mediate induction of c-IAP2 in response to increases in cAMP in T84 cells. We show that cAMP-promoted phosphorylation of CREB and transcriptional activation of c-IAP2 appear in large part to be mediated by activation of ERK1/2 and p38 MAPK, perhaps acting via p90RSK and MSK1. Antibodies and Reagents—Anti-c-IAP2 polyclonal antibody and anti-actin monoclonal antibody (mAb) were from Chemicon International (Temecula, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Polyclonal antibodies against CREB, phospho-CREB, phospho-MSK1, phospho-p90RSK, p44/42 MAPK, phospho-p44/42 MAPK, p38 MAPK, and phospho-p38 MAPK and the MEK1/2 inhibitors PD98059 and U0126 were obtained from Cell Signaling Technology Inc. (San Diego, CA). mAb against Rap1 was from BD Bioscience Pharmingen. Cholera toxin (CTX), 8-CPT-cAMP, forskolin (Fsk), human recombinant TNFα, phorbol 12-myristate 13-acetate, H89, (Rp)-cAMPS, SB203580, and SB202190 were from Calbiochem. PGE2 was from Sigma. 8-CPT-2′-O-Me-cAMP was purchased from BIOLOG Life Science and were purchased from active was purchased from Biotechnology active was a from Chen R. Q. Zhou J.J. S. P. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). to and to were (12Nishihara H. Kizaka-Kondoh S. Insel P.A. Eckmann L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8921-8926Crossref PubMed Scopus (118) Google Scholar). Cell human colon cells were in with and 2 Chinese hamster cells were in with and 2 of and phospho-p38 MAPK, cells were in and of other proteins, cells were in and for were by and analyzed by with the binding was by and by using an cells in were using the with to or to the of a constitutively active promoter as a and CREB, or expression as in the The was with a reporter of was with the T84 cells for were for with or and stimulated with or for were with and in of protein were with 2 of in for was by and by using an ERK1/2 and p38 MAPK of ERK1/2 and p38 MAPK were with a p44/42 MAPK and p38 MAPK Signaling Technology respectively. T84 cells were with inhibitors for by with for were in and for The were with MAPK or MAPK mAb for kinases were with or activating transcription protein for in kinase and phosphorylation of proteins was by with Rap1 were and with inhibitors for by as in the Cell were in and and for The were with of binding domain for and the were with The were by and analyzed by with of cells were with inhibitors for by with or for were stimulated with for and in of cells for were and with the Cell was with a and are as of as in the were by or as with cAMP c-IAP2 Expression through in T84 have that increases in cellular cAMP levels can induce c-IAP2 expression in T84 cells (12Nishihara H. Kizaka-Kondoh S. Insel P.A. Eckmann L. Proc. Natl. Acad. Sci. U. S. 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K. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). directly and increases cAMP We also used CTX, which the of to the activation of and the cAMP of T84 cells with agonists that raise intracellular cAMP increased c-IAP2 promoter in a manner to define the signaling mechanism for the cAMP-promoted increase in c-IAP2 promoter we on the role of PKA, the effector kinase that the of different pharmacological inhibitors of PKA, and showed only inhibition of cAMP-dependent increases in c-IAP2 promoter and protein expression increases in and were inhibited by the of these agents These suggested that c-IAP2 expression is predominantly regulated by pathways in colon cells. were to this cAMP c-IAP2 through of that a in the c-IAP2 promoter is for induction by cAMP (12Nishihara H. Kizaka-Kondoh S. Insel P.A. Eckmann L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8921-8926Crossref PubMed Scopus (118) Google Scholar), suggesting that CREB be a in cAMP-dependent c-IAP2 this we with a c-IAP2 transcriptional reporter that the and is to cAMP (12Nishihara H. Kizaka-Kondoh S. Insel P.A. Eckmann L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8921-8926Crossref PubMed Scopus (118) Google Scholar) and an expression one of of CREB, or CREB forms an with CREB and its to to K.M. J.C. Mol. 6: Google Scholar). CREB a to that phosphorylation of the CREB and its to CRE-dependent transcription Cell. Full Text PDF PubMed Scopus Google Scholar). forms of CREB increased c-IAP2 promoter by and with CREB showed a increase in c-IAP2 promoter with with its CREB was in response to treatment of T84 cells with or 8-CPT-cAMP, and other transcriptional that to CRE, were only these and these that CREB an essential role for c-IAP2 gene transcription in response to increases in CREB by ERK1/2 and p38 of CREB is required for its transcriptional activation B. M. Nat. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Although is an important kinase for CREB we found that in T84 cells the inhibitors and only its cAMP-dependent phosphorylation we that in this other as a kinase of ERK1/2 M. S. Mol. Cell. 1999; PubMed Scopus Google Scholar), and a kinase of ERK1/2 and p38 MAPK M. EMBO J. 1998; 17: PubMed Scopus Google Scholar), of which can CREB J. J.M. E.A. Mol. Cell. Biol. 1998; PubMed Google Scholar), in the phosphorylation of CREB in response to increases in We found that p90RSK and were with and of p90RSK promoted by was inhibited by the MEK1/2 inhibitor U0126 while was but increased by the p38 MAPK inhibitor phosphorylation of was inhibited by U0126 and SB202190 and by the of these agents The inhibitors and or phosphorylation of p90RSK or MSK1. These results that cAMP p90RSK through ERK1/2 and through ERK1/2 and p38 MAPK in T84 cells and that activation of these kinases of CREB phosphorylation promoted by or was significantly by U0126 or SB202190 and this suppression was of of (Rp)-cAMPS, which by only CREB phosphorylation and with U0126 and SB202190 only and inhibition that by the agents and results were obtained by using other inhibitors for ERK1/2 and p38 MAPK, PD98059 and These that ERK1/2 and p38 MAPK are in the cAMP-dependent phosphorylation of cAMP ERK1/2 and p38 MAPK in T84 the results in suggested the involvement of ERK1/2 and p38 MAPK in the phosphorylation of p90RSK and we directly the activation of ERK1/2 and p38 MAPK in response to agents that raise cAMP Treatment of T84 cells with PGE2, Fsk, or phosphorylation of ERK1/2 and p38 MAPK as TNFα as a in kinase assays that increased phosphorylation of ERK1/2 and p38 MAPK was by activation of these or p38 MAPK from cell showed an increased to to or proteins and ERK1/2 kinase was inhibited by treatment with U0126 but was to inhibitors that ERK1/2 activation is in T84 cells. In in cells ERK1/2 activation by cAMP is a inhibited ERK1/2 phosphorylation by a with J.M. de J. M. F. J.L. J.L. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). The inhibitors also to inhibit the cAMP-dependent increase in p38 MAPK these results the that increases in cAMP ERK1/2 and p38 MAPK in T84 and these appear to of In to PKA, recently effector of cAMP is a for the which is to ERK1/2 in cells N. S. S. M. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (118) Google Scholar, J.M. Cell Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). results showed that cAMP-promoted ERK1/2 activation is a we the of a specific of that Rap1 in CHO, and human embryonic kidney cells J.M. de J. M. F. J.L. J.L. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar), to ERK1/2 activation in T84 cells. We found that 8-CPT-2′-O-Me-cAMP to ERK1/2 and p38 MAPK in these cells and as a this was to Rap1 with the that cAMP-promoted ERK1/2 activation in T84 cells via an cAMP c-IAP2 Expression through the of ERK1/2 and p38 the involvement of ERK1/2 and p38 MAPK in cAMP-dependent transcriptional activation of the c-IAP2 of c-IAP2 promoter by and was significantly by the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor and this inhibition was by treatment with inhibitors Consistent with increased c-IAP2 protein expression with and was also inhibited by the of inhibitors of MEK1/2 and p38 MAPK of inhibition of c-IAP2 for the that has a role in cAMP-dependent c-IAP2 an to the role of ERK1/2 and p38 MAPK in the regulation of c-IAP2 we with a c-IAP2 and expression for active forms of MEK1 MKK3, which are of ERK1/2 and p38 MAPK, Chen R. Q. Zhou J.J. S. P. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, J.M. S. 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