A Molecular Link between E2F-1 and the MAPK Cascade
2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês
10.1074/jbc.m610538200
ISSN1083-351X
AutoresJianli Wang, Wen H. Shen, Yan Jin, Paul W. Brandt‐Rauf, Yuxin Yin,
Tópico(s)Ubiquitin and proteasome pathways
ResumoTranscription factor E2F-1 mediates apoptosis and suppresses tumorigenesis. The mechanisms by which E2F-1 functions in these processes are largely unclear. We report here that E2F-1 acts as a transcriptional regulator of MKP-2 (MAPK phosphatase-2), a dual specificity protein phosphatase (DUSP4) with stringent substrate specificity for MAPKs. We show that E2F-1 is required for the cellular apoptotic response to oxidative damage. MKP-2 is greatly increased following oxidative stress, and E2F-1 is necessary for that induction. We found that E2F-1 is physically associated with the MKP-2 promoter and can transactivate the promoter of the MKP-2 gene. Specifically, E2F-1 binds to a perfect palindromic motif in the MKP-2 promoter. Finally, we show that this E2F-1/MKP-2 pathway mediates apoptosis under oxidative stress and that MKP-2 suppresses tumor formation in nude mice. Our findings demonstrate that E2F-1 is a transcriptional activator of MKP-2 and that MKP-2 is an essential cell death mediator in the E2F-1 pathway. Characterization of MKP-2 as a cell death mediator may lead to the development of new strategies for cancer treatment. Transcription factor E2F-1 mediates apoptosis and suppresses tumorigenesis. The mechanisms by which E2F-1 functions in these processes are largely unclear. We report here that E2F-1 acts as a transcriptional regulator of MKP-2 (MAPK phosphatase-2), a dual specificity protein phosphatase (DUSP4) with stringent substrate specificity for MAPKs. We show that E2F-1 is required for the cellular apoptotic response to oxidative damage. MKP-2 is greatly increased following oxidative stress, and E2F-1 is necessary for that induction. We found that E2F-1 is physically associated with the MKP-2 promoter and can transactivate the promoter of the MKP-2 gene. Specifically, E2F-1 binds to a perfect palindromic motif in the MKP-2 promoter. Finally, we show that this E2F-1/MKP-2 pathway mediates apoptosis under oxidative stress and that MKP-2 suppresses tumor formation in nude mice. Our findings demonstrate that E2F-1 is a transcriptional activator of MKP-2 and that MKP-2 is an essential cell death mediator in the E2F-1 pathway. Characterization of MKP-2 as a cell death mediator may lead to the development of new strategies for cancer treatment. Reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; siRNA, small interfering RNA; MEFs, mouse embryo fibroblasts; HMECs, human mammary epithelial cells; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation; TUNEL, terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling. such as superoxide anions, hydrogen peroxide, and hydroxyl radicals are highly relevant to multiple biological processes. High levels of oxidative stress cause damage to multiple cellular components, including DNA, lipid membranes, and protein (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5446) Google Scholar, 2Janssen Y.M. Van Houten B. Borm P.J. Mossman B.T. Lab. Investig. 1993; 69: 261-274PubMed Google Scholar). 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Our previous data show that oxidative stress by H2O2 is a damage signal for the activation of the p53 pathway that leads to apoptosis (11Yin Y. Terauchi T. Solomon G.G. Aizawa S. Rangarajan P.N. Yazaki Y. Kadowaki T. Barrett J.C. Nature. 1998; 391: 707-710Crossref PubMed Scopus (151) Google Scholar, 12Yin Y. Liu Y.X. Jin Y.J. Hall E.J. Barrett J.C. Nature. 2003; 422: 527-531Crossref PubMed Scopus (124) Google Scholar). These studies suggest that ROS may serve both as damage signals that initiate cellular responses and as intermediates for signal transduction important for survival of organisms. Thus, identification of important components involved in the cellular response to oxidative stress may reveal targets for chemotherapeutic strategies. The E2F family includes eight distinct but closely related transcription factors (E2F-1-8) that regulate cell proliferation, development, and apoptosis (13Dyson N. 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This consensus sequence is found in the promoters of a number of genes important for cell cycle progression, including c-myc, cdc2, cdc6, dihydrofolate reductase, thymidine kinase, and the E2F-1 gene itself (21Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar, 22Neuman E. Flemington E.K. Sellers W.R. Kaelin Jr., W.G. Mol. Cell. Biol. 1994; 14: 6607-6615Crossref PubMed Scopus (234) Google Scholar, 23Ohtani K. Tsujimoto A. Ikeda M. Nakamura M. Oncogene. 1998; 17: 1777-1785Crossref PubMed Scopus (91) Google Scholar). E2F-1 plays an important role in both cell cycle regulation (24Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (745) Google Scholar) and apoptosis (25Denchi E.L. Helin K. EMBO Rep. 2005; 6: 661-668Crossref PubMed Scopus (99) Google Scholar). It promotes entry into S phase and stimulates cell cycle progression (26Zhu L. Enders G.H. Wu C.L. Starz M.A. Moberg K.H. Lees J.A. Dyson N. Harlow E. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 75-84Crossref PubMed Scopus (16) Google Scholar). E2F-1 function is regulated by the retinoblastoma protein, which binds to the transactivation domain of E2F-1 and suppresses E2F-1-mediated transcriptional activation (27Helin K. Harlow E. Fattaey A. Mol. Cell. Biol. 1993; 13: 6501-6508Crossref PubMed Scopus (405) Google Scholar, 28Weinberg R.A. Ann. N. Y. Acad. Sci. 1995; 758: 331-338Crossref PubMed Scopus (123) Google Scholar). E2F-1 acts as a cell death mediator in certain settings and functions as a tumor suppressor in a tissue-specific manner (29Weinberg R.A. Cell. 1996; 85: 457-459Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 30Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 31Yamasaki L. Bronson R. Williams B.O. Dyson N.J. Harlow E. Jacks T. Nat. Genet. 1998; 18: 360-364Crossref PubMed Scopus (254) Google Scholar). Overexpression of E2F-1 can trigger apoptosis (32Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (692) Google Scholar, 33Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (810) Google Scholar, 34Kowalik T.F. DeGregori J. Schwarz J.K. Nevins J.R. J. Virol. 1995; 69: 2491-2500Crossref PubMed Google Scholar), indicating that E2F-1 is involved in cell death control. E2F-1 was initially considered to be an oncogene until 1996, when two reports were published on E2F-1 knock-out mice. Mice lacking E2F-1 develop a broad spectrum of tumors (30Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Also, E2F-1-deficient mice exhibit defects in apoptosis and aberrant cell proliferation in some tissues (35Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). These findings demonstrated that E2F-1 is a tumor suppressor. Thus, E2F-1 has two opposing properties: it can stimulate cell cycle progression as an oncogene, or it can promote apoptosis and suppress tumorigenesis as a tumor suppressor gene. The mechanisms by which E2F-1 inhibits tumor progression are unknown. It is commonly believed that E2F-1 functions as a tumor suppressor gene through its induction of apoptosis in p53-dependent and p53-independent manners (36Pan H. Griep A.E. Genes Dev. 1995; 9: 2157-2169Crossref PubMed Scopus (222) Google Scholar). One current focus is on its downstream effectors that signal apoptosis. The most significant target identified so far for E2F-1 function in apoptosis is the p53 homolog p73, which is a tumor suppressor gene (37Oren M. 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Itahana K. Acosta M. Campisi J. Mol. Cell. Biol. 2000; 20: 273-285Crossref PubMed Scopus (341) Google Scholar). In this work, we found that E2F-1 is essential for the cellular response to oxidative stress. We further identified MKP-2 (MAPK phosphatase-2) as a transcriptional target of E2F-1 in this process. MAPKs are key elements in mediating signal transduction from the cell surface to the nucleus (43Guan K.L. Cell. Signal. 1994; 6: 581-589Crossref PubMed Scopus (166) Google Scholar, 44Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (964) Google Scholar). The MAPK cascade is one of the most predominant pathways for cell growth and proliferation (44Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (964) Google Scholar, 45Qi M. Elion E.A. J. Cell Sci. 2005; 118: 3569-3572Crossref PubMed Scopus (421) Google Scholar, 46Reddy K.B. Nabha S.M. Atanaskova N. Cancer Metastasis Rev. 2003; 22: 395-403Crossref PubMed Scopus (466) Google Scholar). MAPKs can be grouped into three families: ERK, JNK, and p38 (47Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (875) Google Scholar). The activity of MAPKs is regulated by dual phosphorylation of their tyrosine and threonine residues. MAPKs are activated upon dual phosphorylation by MAPK/ERK kinase (MEK), which in turn is activated by the Ras/Raf pathway (48Buscher D. Hipskind R.A. Krautwald S. Reimann T. Baccarini M. Mol. Cell. Biol. 1995; 15: 466-475Crossref PubMed Scopus (165) Google Scholar). Upon activation, MAPKs, including ERK1 and ERK2, translocate into the nucleus, where they phosphorylate transduction targets, including transcription factors (49Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar). In contrast, MAPKs are inactivated upon dephosphorylation of tyrosine and threonine residues by MAPK phosphatases (50Dickinson R.J. Keyse S.M. J. Cell Sci. 2006; 119: 4607-4615Crossref PubMed Scopus (279) Google Scholar, 51Theodosiou A. Ashworth A. Genome Biol. 2002; 3 (reviews3009)Crossref PubMed Google Scholar, 52Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1028) Google Scholar, 53Chu Y. Solski P.A. Khosravi-Far R. Der C.J. Kelly K. J. Biol. Chem. 1996; 271: 6497-6501Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). MKP-2 is a dual threonine/tyrosine phosphatase that specifically dephosphorylates and inactivates MAPKs (54Misra-Press A. Rim C.S. Yao H. Roberson M.S. Stork P.J. J. Biol. Chem. 1995; 270: 14587-14596Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). MKP-2 (or HVH2) selectively dephosphorylates the MAPK. Recombinant MKP-2/HVH2 phosphatase exhibits a high substrate specificity toward activated ERK and dephosphorylates both threonine and tyrosine residues of activated ERK1 and ERK2 (55Guan K.L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The mechanism whereby MKP-2 is regulated and its role during tumorigenesis are largely unknown. We found that MKP-2 is transcriptionally regulated by E2F-1. Expression of MKP-2 is increased following oxidative stress, which causes apoptosis in an E2F-1-dependent manner. However, MKP-2 is not responsive to γ-irradiation, which causes DNA damage and leads to cell cycle arrest. Our studies reveal a novel mechanism whereby E2F-1 regulates the expression of MKP-2. Further-more, we found that MKP-2 is an essential target of E2F-1 function in signaling apoptosis following oxidative stress. Our data reveal a molecular link between E2F-1 and the MAPK cascade, which provides insight into the mechanism whereby E2F-1 controls cell death. Cell Lines, Construction of MKP-2 Promoter-Reporters, MKP-2 Expression, and MKP-2 Small Interfering RNA (siRNA) Vectors—E2F-1+/+ and E2F-1-/- primary mouse embryo fibroblasts (MEFs) were isolated from E2F-1+/+ and E2F-1-/- embryos (30Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Mkp-2+/+ and Mkp-2-/- MEFs were purchased from ArtisOptimus Inc. Human MCF-7 breast cancer cells and Saos-2 osteosarcoma cells were from American Type Culture Collection. Human mammary epithelial cells (HMECs) were purchased from Cambrex Bio Science Walkersville, Inc., and were cultured with mammary epithelium basal medium. The MKP-2 promoter was amplified by PCR from human genomic DNA using the following primers. 5′-GAGCGCGGAGGAGCATTAATA-3′ (forward) and 5′-AAACTTGGTCCTCAAGGGCTC-3′(reverse). The PCR product was subcloned into a pT-A vector (Clontech) for sequencing. The promoter was ligated into the pGL3-Basic reporter (Promega), resulting in pGL3/MKP-2-luc. pGL3/MKP-2mt-luc was generated by changing the palindromic sequence from 5′-CTGGCGCCAG-3′ to 5′-CGGGCGCCAG-3′ using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The human MKP-2 expression vector was constructed from a selectable constitutive expression vector, pcDNA3.1 (Invitrogen). The coding region of human MKP-2 was amplified from a human cDNA library by PCR using the following primers: MKP-2-20up1, 5′-GCGTCCCTTCTTAGCTCTCG-3′ (forward); and MKP-2-20dn1, 5′-TGGGAGCCAGCTCTGGTTCT-3′ (reverse). The resulting PCR product (1321 bp) was ligated into pT-Adv and then subcloned into pcDNA3.1 at HindIII and XhoI sites, resulting in pcDNA3/MKP-2. An E2F-1 siRNA expression vector (U6/siE2F-1) was constructed as described previously (56Shen W.H. Yin Y. Broussard S.R. McCusker R.H. Freund G.G. Dantzer R. Kelley K.W. J. Biol. Chem. 2004; 279: 7438-7446Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The following primers were used to generate the MKP-2 siRNA vector: 5′-CACCGACTACCCCGGACCCTTCAAGAGAGGGTCCGGGGTAGTCGGTGTTTTTT-3′ (forward) and 5′-AATTAAAAAACACCGACTACCCCGGACCCTCTCTTGAAGGGTCCGGGGTAGTCGGTGGGCC-3′ (reverse). A random sequence from human intracellular interleukin-1 receptor agonist noncoding sequence was ligated into U6 as a scrambled control. The constructs were sequenced to confirm that the designed fragments were introduced in-frame into vectors. The annealed oligonucleotides were cloned into the designed sites of ApaI and EcoRI in the pSilencer 1.0-U6 siRNA expression vector (Ambion, Inc.). Gene Transfection and Luciferase Assay—Cells were transiently transfected with plasmids using Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. Luciferase reporters were transfected into MCF-7 cells along with a pCMV-β-gal reporter plasmid to normalize the transfection efficiency. Cell extracts were processed using a Dual-Light kit (Tropix) according to the manufacturer's instructions. Luciferase activity was measured with a Berthold Autolumat LB953 rack luminometer. Luciferase values were normalized against β-galactosidase activity. RNA Isolation and Northern and Western Blotting—Total RNA was isolated from growing cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For Northern analysis, RNA was run on a 1.2% formaldehyde gel and transferred to a HN membrane using a TurboBlotter system (Schleicher & Schüll). DNA probes were labeled with [α-32P]dCTP (Amersham Biosciences) using a Prime-It RmT random primer labeling kit (Stratagene). The membrane was hybridized with labeled DNA probes in QuikHyb hybridization solution (Stratagene) at 65 °C for 2 h and developed for autoradiography. For Western blotting, growing cells were lysed in cold Nonidet P-40 buffer with protease/phosphatase inhibitors. The samples were resolved on 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose filters. The blots were incubated with primary antibodies, followed by incubation with peroxidase-conjugated secondary antibodies. The signals were detected by enhanced chemiluminescence (Amersham Biosciences). Electrophoretic Mobility Shift Assay—Oligonucleotides were annealed and labeled with 32P using T4 polynucleotide kinase and [γ-32P]ATP as described (57Maiyar A.C. Huang A.J. Phu P.T. Cha H.H. Firestone G.L. J. Biol. Chem. 1996; 271: 12414-12422Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Human GST-E2F-1 fusion protein was produced from growing Escherichia coli BL21 cells transfected with pGEX-2T-E2F-1 and induced with isopropyl β-d-thiogalactopyranoside (58Karlseder J. Rotheneder H. Wintersberger E. Mol. Cell. Biol. 1996; 16: 1659-1667Crossref PubMed Scopus (315) Google Scholar). The E2F-1 fusion protein was purified by affinity chromatography. Electrophoretic mobility shift assay was conducted as described previously (12Yin Y. Liu Y.X. Jin Y.J. Hall E.J. Barrett J.C. Nature. 2003; 422: 527-531Crossref PubMed Scopus (124) Google Scholar). Briefly, 32P-labeled probes were mixed with 0.5 μg of purified recombinant GST-E2F-1 fusion protein in 20 μl of DNA binding reaction buffer consisting of 20 mm Tris-HCl (pH 7.5), 4% Ficoll 400, 2 mm EDTA, 0.5 mm dithiothreitol, and 0.2 mg of poly(dI-dC). For supershift detection, an anti-E2F-1 monoclonal anti-body (Santa Cruz Biotechnology, Inc.) was included in reactions. The reaction mixtures were incubated at 4 °C for 20 min, resolved on a 4% polyacrylamide gel, and exposed for autoradiography. Chromatin Immunoprecipitation (ChIP)—Growing cells were harvested and processed for ChIP using the anti-E2F-1 antibody and a ChIP assay kit (Upstate) according to the manufacturer's protocol. Briefly, cells were cross-linked with 1% formaldehyde, and chromatin was sonicated to 500-2000-bp fragments. Precleared lysates were incubated with the anti-E2F-1 monoclonal antibody. The chromatin samples without addition of the antibody were used as negative controls, and genomic DNA was used as a positive control for PCR (input). Immune complexes were precipitated with protein A beads; protein-DNA cross-links were reversed by boiling for 30 min in 100 mm Tris (pH 6.8), 10% β-mercaptoethanol, and 4% SDS; and DNA was phenol-extracted and ethanol-precipitated. The precipitated MKP-2 promoter was amplified by PCR using the primers spanning the potential E2F-1-binding site. E2F-1 Is Necessary for the Cellular Response to Oxidative Stress—A wide variety of mutagenic agents are toxic to cells, but the mechanisms of toxicity can vary. Some agents irreversibly induce growth arrest, whereas others induce cell death by apoptosis or necrosis. For example, γ-radiation causes cell cycle arrest in MEFs (59Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2316) Google Scholar, 60Kastan M.B. Zhan Q. El Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2931) Google Scholar), whereas oxidative stress induces cell death by apoptosis in the same cells (61Yin Y. Solomon G. Deng C. Barrett J.C. Mol. Carcinog. 1999; 24: 15-24Crossref PubMed Scopus (64) Google Scholar). We demonstrated previously that p53 is required for the cellular response to oxidative stress, leading to cell death (11Yin Y. Terauchi T. Solomon G.G. Aizawa S. Rangarajan P.N. Yazaki Y. Kadowaki T. Barrett J.C. Nature. 1998; 391: 707-710Crossref PubMed Scopus (151) Google Scholar). Because E2F-1 plays an important role in the induction of apoptosis and because E2F-1 cooperates with p53 in mediating apoptosis in some cell types (32Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (692) Google Scholar, 33Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (810) Google Scholar), it is possible that E2F-1 is also involved in the cellular defense against oxidative damage. To determine whether E2F-1 is necessary for the cellular response to oxidative stress, we chose an E2F-1 knock-out system (E2F-1+/+ and E2F-1-/- MEFs) and tested the susceptibility of these cells to oxidative damage. As shown in Fig. 1A, E2F-1+/+ MEFs were extremely sensitive to H2O2 treatment. Most E2F-1+/+ MEFs were rapidly killed by H2O2. In contrast, E2F-1-/- MEFs were highly resistant to cell killing by H2O2. To determine the nature of oxidative cell death, we performed a TUNEL assay to identify apoptotic cells in MEF population. As shown in Fig. 1B, the majority of E2F-1+/+ MEFs were killed under oxidative stress through typical apoptosis because the apoptotic cells were stained as double-colored cells by the TUNEL assay (panel a versus panel b). In contrast, a much lower percentage of E2F-1-/- MEFs was stained as apoptotic cells following oxidative damage by H2O2 (panel c versus panel d). These data suggest that E2F-1 is necessary for the cellular response to cell death induction following oxidative stress. To determine whether E2F-1 mediates cell death by oxidative damage, we established a cell system in which E2F-1 is ectopically expressed. We introduced the cytomegalovirus-driven human E2F-1 expression vector (pCMV/E2F-1) into a breast cancer cell line, MCF-7. We obtained stable clones expressing ectopic E2F-1 through G418 selection and confirmed the expression of E2F-1 protein by immunoblotting using the anti-E2F-1 antibody. These clones were designated as MCF-7/E2F-1, followed by clone number, such as MCF-7/E2F-1-C3, -C5, and -C8 (Fig. 1C). To test whether these cells expressing E2F-1 are sensitive to oxidative damage, we treated these clones and MCF-7/pCMV cells as a control with H2O2 for different times. As shown in Fig. 1D, although MCF-7/pCMV cells were resistant to cell killing by H2O2, MCF-7/E2F-1 cells were highly susceptible to treatment with H2O2. The apoptotic process was extensive, and most cells were killed within 24 h of H2O2 treatment (Fig. 1D). To avoid the interference of clonal variation, we also obtained three clones of MCF-7/pcDNA3 cells and did not find significantly different sensitivity of these cell clones to H2O2-induced cell death compared with parental MCF-7 cells. These results indicate that E2F-1 effectively mediates cell death following oxidative damage. Transcriptional Regulation of MKP-2 by E2F-1 in Response to Oxidative Stress—Transcription factors execute their function by regulating downstream target genes. To identify potential targets of E2F-1 in cell death induction, we utilized GeneChip technology (62Zhao R. Gish K. Murphy M. Yin Y. Notterman D. Hoffman W.H. Tom E. Mack D.H. Levine A.J. Genes Dev. 2000; 14: 981-993Crossref PubMed Scopus (277) Google Scholar) to analyze the gene expression profiles in MEFs with or without E2F-1 (E2F-1+/+ and E2F-1-/- MEFs) under normal and stress conditions. MKP-2 was among the genes responsive to oxidative stress in E2F-1+/+ MEFs, but not in E2F-1-/- MEFs (data not shown). To confirm the data from the microarray readout, we performed Northern and Western analyses of MKP-2 expression in the same cell model. E2F-1+/+ and E2F-1-/- MEFs were either exposed to γ-irradiation, which causes cell cycle arrest at G1 (60Kastan M.B. Zhan Q. El Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2931) Google Scholar), or treated with H2O2, which induces cell death by apoptosis (61Yin Y. Solomon G. Deng C. Barrett J.C. Mol. Carcinog. 1999; 24: 15-24Crossref PubMed Scopus (64) Google Scholar). As shown in Fig. 2 (A and B), MKP-2 was low in E2F-1+/+ MEFs and not induced by γ-irradiation (lanes 1 and 2). However, MKP-2 was markedly increased following oxidative damage by H2O2 (lane 3 versus lane 1). Furthermore, the transcriptional response of MKP-2 to oxidative damage was ablated in E2F-1-/- MEFs (lane 6 versus lane 3). These observations suggest that MKP-2 is inducible by oxidative stress and that E2F-1 is required for induction of MKP-2 in response to oxidative damage. It is well known that MAPK family members, including ERK, JNK, and p38, are involved in apoptosis (63Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar). Because MKP-2 is an inhibitor of MAPKs, it is possible that E2F-1 can also influence these MAPKs following oxidative stress. Thus, we examined the relationship between the phosphorylation status of ERK1/2, JNK, and p38 and the status of E2F-1. As shown in Fig. 2C, the level of ERK1/2 phosphorylation was largely reduced in E2F-1+/+ MEFs upon oxidative damage (lane 2) but remained unchanged in E2F-1-/- MEFs upon the same treatment (lane 4). Although p38 was slightly dephosphorylated following oxidative damage (Fig. 2D, lane 2), this change was independent of E2F-1 status because p38 was dephosphorylated in both E2F-1+/+ and E2F-1-/- MEFs (lanes 2 and 4). In contrast, JNK was phosphorylated following oxidative damage in an E2F-1-independent manner (middle panel). These results indicate that ERK1, but not p38 and JNK, is the major target of E2F-1 signaling. It has been reported that MKP-2 selectively dephosphorylates the MAPK ERK1/2 (55Guan K.L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), and our data are consistent with this observation. To determine whether E2F-1 regulates the expression of MKP-2 and suppresses the activity of the MAPK ERK1/2, we chose the MCF-7/E2F-1 cell system described for Fig. 1C. As shown in Fig. 2E, the MKP-2 transcript was low in MCF-7/pCMV cells (lane 1). However, MKP-2 transcription was increased in MCF-7/E2F-1-C3, -C5, and -C8 (lanes 2-4). Correspondingly, the levels of MKP-2 protein were also increased by E2F-1 (Fig. 2F, lanes 2-4). These results suggest that MKP-2 transcription is up-regulated by E2F-1 in MCF-7 cells. MKP-2 can inactivate ERK1 and ERK2 kinase activity through dephosphorylation of
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