Portal de Periódicos da CAPES

E2F1 Modulates p38 MAPK Phosphorylation via Transcriptional Regulation of ASK1 and Wip1

2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês

10.1016/s0021-9258(19)84043-7

1083-351X

Tzippi Hershko, Katya Korotayev, Shirley Polager, Doron Ginsberg,

Virus-based gene therapy research

The E2F family of transcription factors regulates a diverse array of cellular functions, including cell proliferation, cell differentiation, and apoptosis. Recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways. We show here that E2F1 modulates the activity of the p38 MAPK pathway via E2F1-induced transient up-regulation of p38 MAPK phosphorylation. The mechanism by which E2F1 modulates p38 MAPK phosphorylation involves transcriptional induction of the kinase ASK1, a member of the MAPKKK family that phosphorylates p38 MKKs. Subsequent E2F-dependent down-regulation of the p38 signaling pathway is achieved through E2F-induced up-regulation of Wip1, a phosphatase that dephosphorylates and inactivates p38. Both ASK1 and Wip1 are essential mediators of the E2F-p38 connection: knock down of ASK1 inhibits E2F1-induced phosphorylation of p38, whereas knock down of Wip1 prolongs E2F1-induced p38 phosphorylation. Furthermore, Wip1 knock down enhances E2F1-induced apoptosis. Therefore, our data reveal a novel link between a central signaling pathway and the transcription factor E2F and identify Wip1 as a modulator of E2F1-induced apoptosis. The E2F family of transcription factors regulates a diverse array of cellular functions, including cell proliferation, cell differentiation, and apoptosis. Recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways. We show here that E2F1 modulates the activity of the p38 MAPK pathway via E2F1-induced transient up-regulation of p38 MAPK phosphorylation. The mechanism by which E2F1 modulates p38 MAPK phosphorylation involves transcriptional induction of the kinase ASK1, a member of the MAPKKK family that phosphorylates p38 MKKs. Subsequent E2F-dependent down-regulation of the p38 signaling pathway is achieved through E2F-induced up-regulation of Wip1, a phosphatase that dephosphorylates and inactivates p38. Both ASK1 and Wip1 are essential mediators of the E2F-p38 connection: knock down of ASK1 inhibits E2F1-induced phosphorylation of p38, whereas knock down of Wip1 prolongs E2F1-induced p38 phosphorylation. Furthermore, Wip1 knock down enhances E2F1-induced apoptosis. Therefore, our data reveal a novel link between a central signaling pathway and the transcription factor E2F and identify Wip1 as a modulator of E2F1-induced apoptosis. The E2F family of transcription factors comprises eight structurally related E2Fs (E2F1–8), most of which function as heterodimers with members of the DP family. E2F transcriptional activity is negatively regulated by the product of the retinoblastoma (RB) 4The abbreviations used are: RB, retinoblastoma; OHT, 4-hydroxytamoxifen; RT-PCR, reverse transcription PCR; ChiP, chromatin immunoprecipitation; siRNA, small interference RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAP kinase kinase; MAPKKK or MAP3K, MAPK kinase kinase; ASK1, apoptosis signal-regulating kinase 1; ER, estrogen receptor; ATM, ataxia-telangiectasia mutated. 4The abbreviations used are: RB, retinoblastoma; OHT, 4-hydroxytamoxifen; RT-PCR, reverse transcription PCR; ChiP, chromatin immunoprecipitation; siRNA, small interference RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAP kinase kinase; MAPKKK or MAP3K, MAPK kinase kinase; ASK1, apoptosis signal-regulating kinase 1; ER, estrogen receptor; ATM, ataxia-telangiectasia mutated. tumor suppressor pRB (1DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar), and the growth suppression activity of RB is dependent on its ability to interact with E2F. The RB pathway is often mutated in human cancer, resulting in deregulated activity of E2F (2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar). E2Fs are best known for their involvement in the timely activation of genes required for cell cycle progression (1DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar). However, it is currently clear that the E2F family plays a major role in regulating a diverse array of cellular functions, including cell proliferation, cell differentiation, and apoptosis. Recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways (3Chaussepied M. Ginsberg D. Mol. Cell. 2004; 16: 831-837Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 4Chaussepied M. Ginsberg D. Cell Cycle. 2005; 4Crossref PubMed Scopus (13) Google Scholar). First, many components of such pathways are up-regulated in screens that analyze changes in gene expression in response to E2F (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (630) Google Scholar, 6Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (496) Google Scholar, 7Ma Y. Croxton R. Moorer Jr., R.L. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (96) Google Scholar, 8Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (222) Google Scholar, 9Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Crossref PubMed Scopus (909) Google Scholar, 10Young A.P. Nagarajan R. Longmore G.D. Oncogene. 2003; 22: 7209-7217Crossref PubMed Scopus (73) Google Scholar). Second, studies of particular signaling pathways demonstrated that E2F affects the level and activity of particular components in these pathways. For example, activation of the pRB/E2F pathway sensitized fibroblasts to basic fibroblast growth factor, most probably due to transcriptional up-regulation of fibroblast growth factor receptor 1 by E2F (11Tashiro E. Maruki H. Minato Y. Doki Y. Weinstein I.B. Imoto M. Cancer Res. 2003; 63: 424-431PubMed Google Scholar). In addition, E2F was recently shown to positively affect the phosphatidylinositol 3-kinase/AKT signaling pathway through the transcriptional induction of the adaptor protein Gab2 (3Chaussepied M. Ginsberg D. Mol. Cell. 2004; 16: 831-837Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, a number of studies suggest that E2F can up-regulate the levels of the MAPKKK ASK1 (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (630) Google Scholar, 12Stanelle J. Stiewe T. Theseling C.C. Peter M. Putzer B.M. Nucleic Acids Res. 2002; 30: 1859-1867Crossref PubMed Scopus (146) Google Scholar); however, the functional consequence of this elevation is not fully understood. In an attempt to further establish a functional link between E2F1 and major signaling pathways we tested the effects of E2F1 on the MAPK p38 pathway. The p38 MAPK signaling pathway plays an essential role in regulating many cellular processes, including inflammation, cell differentiation, and cell growth and death. A variety of signaling events such as inflammatory stimuli, genotoxic stresses, and growth factors are able to trigger the activation of the p38 pathway (13Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1389) Google Scholar, 14Engelberg D. Semin. Cancer Biol. 2004; 14: 271-282Crossref PubMed Scopus (88) Google Scholar, 15Saklatvala J. Curr. Opin. Pharmacol. 2004; 4: 372-377Crossref PubMed Scopus (374) Google Scholar). Like all MAP kinase cascades, the p38 cascade consists of three classes of serine/threonine kinases, MAP kinase, MAPK kinase (MAPKK), and MAPKK kinase (MAP3K). MAP3K phosphorylates and thereby activates MAPKK, and activated MAPKK in turn phosphorylates and activates MAP kinase. Several MAP3Ks can induce p38 activation. These include ASK1/MAPKKK5, MTK1, MLK2/MST, MLK3/PTK/SPRK, DLK/MUK/ZPK, and TAK1 (13Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1389) Google Scholar). Overexpression of these MAP3Ks leads to activation of both p38 and JNK pathways. Under physiological conditions, MAP kinase activation is often transient, and dephosphorylation by phosphatases seems to play a major role in the down-regulation of MAP kinase activity. Different phosphatases function at different levels to inactivate the MAP kinase cascades, and several of these phosphatases were found to be regulated by transcriptional control. In studying the regulation of the p38 signaling pathway by E2F, we focused on a MAPKKK that activates this pathway, ASK1 (16Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2017) Google Scholar, 17Matsukawa J. Matsuzawa A. Takeda K. Ichijo H. J. Biochem. (Tokyo). 2004; 136: 261-265Crossref PubMed Scopus (275) Google Scholar, 18Sumbayev V.V. Yasinska I.M. Arch. Biochem. Biophys. 2005; 436: 406-412Crossref PubMed Scopus (111) Google Scholar), and a phosphatase that inactivates this pathway, Wip1 (19Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (357) Google Scholar). Apoptosis signal-regulating kinase 1 (ASK1)/MAP3K5 is a member of the MAPKKK family that activates both JNK and p38 MAP kinase pathways by a direct site-specific Ser/Thr phosphorylation of their respective MKKs, MKK3/MKK6 for p38 kinases and MKK4/MKK7 for JNK (16Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2017) Google Scholar). The protein phosphatase Wip1 is a member of the serine/threonine-specific protein phosphatase type 2C family (19Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (357) Google Scholar, 20Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (458) Google Scholar). In vivo and in vitro studies indicate that Wip1 selectively dephosphorylates and inactivates p38, but not JNK, ERK, or MAPKKS (19Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (357) Google Scholar). We show here that E2F activation leads to a transient increase in p38 phosphorylation through a transcription-dependent mechanism. We demonstrate that ASK1 is an E2F target gene and an essential effector of E2F-dependent p38 activation. In addition, we identify the phosphatase Wip1 as a new E2F target gene that is essential for E2F-dependent down-regulation of the p38 signaling pathway. Furthermore, we show that Wip1 inhibits E2F1-induced apoptosis, suggesting the existence of an E2F1-Wip1 negative feedback loop. These results reveal a functional link between a central signaling pathway and the transcription factor E2F. Cell Culture—U2OS osteosarcoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum. Early passage WI38 human embryonic lung fibroblasts were grown in minimal essential medium supplemented with 15% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, and non-essential amino acids. H1299 lung adenocarcinoma cells were cultured in RPMI 1640 supplemented with 5% fetal calf serum. Cells were maintained at 37 °C in a humidified 8% CO2-containing atmosphere. To induce activation of ER-E2F-1, cells were treated with 4-hydroxytamoxifen (OHT) (300 nm) for the time indicated. Cycloheximide (Sigma) was used for 4 h, 10 μg/ml. RT-PCR—Total RNA was extracted from the cells using the RNeasy kit (Qiagen). Reverse transcription-PCR (RT-PCR) was performed as previously described (8Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (222) Google Scholar). Primers for ASK1 were: 5′-ACAGCAGATACTCTCAGCC and 5′-CATTGTCACCCTTTATGTCCC. Primers for Wip1 were: 5′-TTCTCGCTTGTCACCTTGCC and 5′-CCAAACTACACGATTCACCCC. Primers for GAPDH were: 5′-ACCACAGTCCATGCCATCAC and 5′-TCCACCACCCTGTTGCTGTA. Western Blotting—Cells were lysed in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40) in the presence of protease inhibitor mixture (Complete mini; Roche Applied Science) and phosphatase inhibitor mixtures I and II (P2850, P5726; Sigma). Equal amounts of protein from each lysate, as determined by Bradford assay, were resolved by electrophoresis in an SDS 10% polyacrylamide gel and then transferred to a membrane (Protran BA 85, S&S). Membrane was incubated overnight with the following antibodies: anti-Wip1 antibody (2380-MC-100; R&D Systems), anti-phospho-p38 (AF869; R&D Systems), anti-E2F1 (sc-251; Santa Cruz Biotechnology), anti-GAPDH (Chemicon), anti-p38 (gift from Prof. Roni Zeger, Weizmann institute, Israel), anti-ASK1 (sc-7931; Santa Cruz), and anti-phospho-p53 (Ser-46) (2521; Cell Signaling). Binding of the primary antibody was detected using an enhanced chemiluminescence kit (ECL; Amersham Biosciences). Plasmids—The following plasmids have been described previously: pBabe-puro-HA-ER-E2F1 and pBabe-puro-HA-ER-E2F1 E132 (21Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Muller H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (531) Google Scholar), pRetroSuper p53siRNA (22Berkovich E. Ginsberg D. Oncogene. 2003; 22: 161-167Crossref PubMed Scopus (100) Google Scholar). pBabe-puro-HA-ER-E2F2, pBabe-puro-HA-ER-E2F3, and pBabe-puro-E1A12S were kind gifts from K. Helin. Short hairpin RNAs specific for human ASK1 (ASK1 5′-GGAGTATGACTATGAATAT and ASK2 5′-GTTACTTTCCTACAGAGATA), and Wip1 (Wip1 5′-GCGAAAGAACTCTGTTAAA and Wip2 5′-GAAGAAGCATAGACGAAAT) were cloned into the retroviral pRETROSUPER vector (23Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar). Transfection and Infection Assays—Cells of the packaging cell line 293T (2 × 106cells) were co-transfected with 10 μgof ψ ecotropic packaging plasmid, pSV-ψ-E-MLV, providing packaging helper function and 10 μg of the relevant plasmid using the calcium phosphate method in the presence of Chloroquin (25 μm final concentration, C6628; Sigma). After 8 h, the transfection medium was replaced with fresh Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, and 5-ml aliquots of cell supernatant containing retroviruses were collected at 6-h intervals. Five collections were pooled together and frozen in aliquots. For infection the cells were incubated for 5 h at 37 °C in 3 ml of retroviral supernatant supplemented with 8 μg/ml of polybrene (H9268; Sigma). Then, 7 ml of medium was added, and after 24 h the medium was replaced with fresh medium containing 2 μg/ml of puromycin (P7130; Sigma). Chromatin Immunoprecipitation—Chromatin immunoprecipitation was performed essentially as described (24Hershko T. Ginsberg D. J. Biol. Chem. 2004; 279: 8627-8634Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Antibodies to E2F1 (sc-193; Santa Cruz), E2F2 (Sc-633; Santa Cruz), E2F3 (sc-879; Santa Cruz), E2F4 (sc-1082+ sc-866; Santa Cruz), and p53 (CM1; Novocastra) (1.2 μg/immunoprecipitation) were used to precipitate chromosomal DNA, employing cross-linked chromatin prepared from exponentially growing cells. The immunoprecipitated DNA was analyzed by PCR using the following primers: ASK1 5′-GAGTGGGTGGCCAGAAGC and 5′-CGGAGCTTCCTTTTCTTGGC, Wip1 5′-ATCCAGCTCAAGGGTGCAGC and 5′-AGCAGAGCCAGGCAGTTGCG, and GAPDH, 5′-GTATTCCCCCAGGTTTACAT and 5′-TTCTGTCTTCCACTCACTCC. Fluorescence-activated Cell Sorter Analysis—Cells were trypsinized and fixed with 70% ethanol (4 °C, overnight). After fixation, cells were centrifuged for 4 min at 1500 rpm and incubated for 30 min at 4 °C in 1 ml of phosphate-buffered saline, centrifuged, and resuspended in phosphate-buffered saline containing 5 mg/ml propidium iodide and 50 μg/ml RNase A for 20 min at room temperature. Fluorescence intensity was analyzed using a BD Biosciences flow cytometer. In response to a variety of genotoxic stresses, including UV and γ radiation, p38 MAP kinase undergoes rapid phosphorylation on Thr-180 and Tyr-182. These phosphorylations lead to activation of the p38 kinase. To test the effect of E2F1 on phosphorylation and activation of p38, we expressed a conditionally active E2F1 (ER-E2F1) in WI38 human embryonic lung fibroblasts and U2OS human osteosarcoma cells and analyzed the effect of E2F1 activation on p38 phosphorylation on Thr-180 and Tyr-182. The ER-E2F-1 is expressed as an inactive fusion protein in the cytoplasm, and upon addition of the OHT it translocates to the nucleus and transactivates E2F-regulated genes (25Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (283) Google Scholar). Activation of E2F1 in WI38 cells by addition of OHT did not affect the protein levels of p38 (Fig. 1A, lower panel). However, it led to an increase in the phosphorylation level of p38 in a transient fashion; phosphorylation of p38 was detected 8 h after E2F1 activation, persisted till 12 h after E2F1 activation, and then declined (Fig. 1A). Similar results were obtained using U2OS cells (data not shown). Activation of a DNA binding-deficient mutant of E2F1 (E2F1-E132) failed to induce p38 phosphorylation, indicating that the effect of E2F1 on p38 activation occurs through a transcription-dependent mechanism. (Fig. 1A). Similarly, expression of E2F1 in U2OS cells resulted in a significant increase in the levels of phosphorylated p38 (Fig. 1B). Introduction of E2F1-E132 did not affect the levels of phosphorylated p38 (Fig. 1B), further demonstrating that the effect of E2F1 on p38 phosphorylation is transcription dependent. Recent screens employing DNA microarrays demonstrated that ectopic expression of E2F1 elevates the levels of ASK1, a member of the MAPKKK family that can activate the p38 pathway (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (630) Google Scholar, 12Stanelle J. Stiewe T. Theseling C.C. Peter M. Putzer B.M. Nucleic Acids Res. 2002; 30: 1859-1867Crossref PubMed Scopus (146) Google Scholar, 26Jamshidi-Parsian A. Dong Y. Zheng X. Zhou H.S. Zacharias W. McMasters K.M. Gene. 2005; 344: 67-77Crossref PubMed Scopus (21) Google Scholar). Therefore, we further characterized the regulation of ASK1 by E2F and its possible involvement in E2F1-induced p38 phosphorylation. Introduction of WT E2F1 into U2OS cells resulted in a significant increase in the endogenous mRNA levels of ASK1 (Fig. 2A). Introduction of a DNA binding-deficient mutant of E2F1 failed to increase ASK1 mRNA levels (Fig. 2A), as was shown for p38 phosphorylation (Fig. 1). Activation of conditionally active E2F1 as well as E2F2 or E2F3 in U2OS also led to an increase in mRNA levels of ASK1 (Fig. 2, B and C). The E2F1-induced increase in ASK1 mRNA was detected also in the presence of the protein synthesis inhibitor cycloheximide (Fig. 2C). Activation of ER-E2F-1 does not require de novo protein synthesis (25Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (283) Google Scholar), and therefore this increase in the presence of cycloheximide indicates that de novo protein synthesis is not required for E2F1-mediated up-regulation of ASK1 mRNA, suggesting that the ASK1 gene is a direct E2F target. Examination of the human genomic sequence spanning 1000 base pairs upstream of the transcription start site of the ASK1 gene revealed the presence of a putative E2F binding site at position –608/–615 (GCGGGGAA) from the transcription start site. Chromatin Immunoprecipitation (ChIP) analysis of the human ASK1 upstream region demonstrated that endogenous E2F3, E2F4, and to a lesser extent E2F1 and E2F2, bind this promoter in WI38 cells (Fig. 2D). No binding was detected when using an anti-p53 antibody as a control (Fig. 2D). Similarly, ChIP analysis using U2OS cells containing ER-E2F1 demonstrated that E2F1 binds this promoter and this binding is enhanced after activation of ER-E2F1 (Fig. 3E). No such enhancement was detected when the GAPDH promoter was studied (data not shown). E2F1 activates p38 in a transient fashion (Fig. 1A). One possible explanation for the observed decrease in E2F1-induced p38 phosphorylation, following the initial increase, is that E2F up-regulates the expression and activity of a p38 phosphatase. One documented p38 phosphatase is Wip1 (also named PPM1D), which is a member of the serine/threonine-specific protein phosphatase type 2C family (20Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (458) Google Scholar). We therefore tested whether Wip1 is a novel E2F target gene whose up-regulation is responsible for the transient nature of the effect of E2F1 on p38 phosphorylation. Ectopic expression of WT E2F1, but not the E2F1-E132 DNA binding-deficient mutant, in U2OS cells led to an increase in endogenous Wip1 mRNA (Fig. 3A). Consistently, activation of ER-E2F1 up-regulated both mRNA and protein levels of endogenous Wip1 (Fig. 3, B and C). The increase in Wip1 mRNA after activation of ER-E2F1 was detected also in the presence of the protein synthesis inhibitor cycloheximide (Fig. 3C), suggesting that Wip1 is a direct E2F target. Examination of the human genomic sequence spanning 1000 base pairs upstream of the transcription start site of the Wip1 gene revealed the presence of a putative E2F binding site at position –423/–430 (TTTCCCGC) from the transcription start site. ChIP analysis of the human Wip1 upstream region spanning from –491 to +48 and encompassing the putative E2F binding site demonstrated that endogenous E2F1 and E2F4 bind this promoter in WI38 cells (Fig. 3D). Similarly, ChIP analysis using U2OS cells containing ER-E2F1 demonstrated that E2F1 binds this promoter and this binding is enhanced after activation of ER-E2F1 (Fig. 3E). Binding of E2F4 to the promoters of ASK1 and Wip1 probably reflects the fact that chromatin was prepared from unsynchronized cell culture in which >50% of the cells were in G1 and that E2F4, the predominant E2F family member, is bound to many E2F-responsive genes in early to mid G1 (27Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar). Because Wip1 is a p53-inducible phosphatase (20Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (458) Google Scholar), we studied the possible involvement of p53 in E2F1-induced up-regulation of Wip1. Activation of ER-E2F1 in the p53-deficient human H1299 non-small cell lung carcinoma cells resulted in elevated levels of Wip1 mRNA (Fig. 4A). In addition, ectopic expression of the adenoviral oncoprotein E1a, which disrupts RB-E2F complexes thereby leading to deregulated endogenous E2F activity, also led to an increase of Wip1 mRNA in H1299 cells (Fig. 4B). An involvement of p53 in the E2F1-induced up-regulation of Wip1 was further excluded by the finding that activation of ER-E2F1 led to increased Wip1 mRNA levels in the presence of siRNA that abolished human p53 expression in U2OS cells (Fig. 4C). Taken together, these data indicate that regulation of Wip1 by E2F1 is p53 independent. These data are in agreement with the demonstration that E2F1-induced up-regulation of Wip1 does not require de novo protein synthesis (Fig. 3C) and also with the binding of endogenous E2F1 and E2F4 to the Wip1 promoter (Fig. 3D). Furthermore, the up-regulation of Wip1 mRNA by E1a, taken together with the ChIP data, demonstrates that Wip1 is a target for endogenous E2F activity. Our data show that E2F1 affects the levels of a kinase and a phosphatase that modulate p38 activity. The relative strength and kinetics with which ASK1 and Wip1 respond to E2F activity may determine a window of time in which p38 is active. This will be possible if activation of E2F1 leads first to up-regulation of ASK1 that mediates p38 phosphorylation and only later E2F1 up-regulates the phosphatase Wip1 that dephosphorylates and inactivates p38. To test this hypothesis, we studied the kinetics of ASK1 and Wip1 up-regulation upon activation of E2F1. As can be seen in Fig. 5, protein level of ASK1 was already increased 8 h after E2F1 activation whereas elevation of Wip1 protein level was detected only 13 h after E2F1 activation. These data imply that E2F1 regulates the expression of these two genes differentially and level of ASK1 protein is increased prior to that of Wip1. Consistent with the kinetics of elevation of ASK1 and Wip1 protein levels, phosphorylation of p38 was detected 8 h after E2F1 activation, persisted until 13 h after E2F1 activation, and then declined (Figs. 1 and 5). Moreover, E2F1 activation led to a transient increase in phosphorylation of p53 on serine 46, a known phosphorylation site of p38 (28Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (594) Google Scholar), demonstrating that E2F1 up-regulates p38 activity (Fig. 5). Interestingly, in this experiment the decrease in phospho-p38 levels at late time points was not back to basal levels even though Wip1 levels were high. This raises the possibility that Wip1 is not the only protein regulating p38 dephosphorylation. To assess the relevance of ASK1 for the E2F-mediated activation of p38, we tested the effect of reducing ASK1 expression on E2F1-induced p38 phosphorylation. To that end, U2OS cells expressing ER-E2F1 were infected with retroviruses expressing either ASK1-specific siRNA or irrelevant siRNA. Two distinct ASK1-specific siRNAs were used, and expression of each one of them significantly impaired E2F1-induced ASK1 expression (Fig. 6A). Importantly, interfering with ASK1 induction significantly impaired p38 phosphorylation in response to E2F1 activation (Fig. 6, B and C). These data indicate that E2F1-mediated up-regulation of ASK1 contributes to E2F1-induced phosphorylation of p38. To assess the role of Wip1 in the effects of E2F1 on p38 phosphorylation, we examined the effect of reducing Wip1 expression on the extent and duration of E2F1-induced p38 phosphorylation. To that end, U2OS cells expressing ER-E2F1 were infected with retroviruses expressing either Wip1-specific siRNA or irrelevant siRNA. Again, two distinct siRNAs directed against Wip1 were used, and expression of each one of them significantly inhibited and delayed E2F1-induced expression of Wip1 (Fig. 7, A and B, lower panels). Expression of the two siRNAs that inhibit Wip1 expression, but not an irrelevant siRNA, inhibited and delayed E2F1-induced p38 dephosphorylation (Fig. 7A, upper panel, compare lanes 3 and 4 to lanes 7 and 8, and Fig. 7B, upper panel, compare lanes 3 and 7). These results indicate that E2F1-mediated up-regulation of Wip1 contributes to E2F1-induced dephosphorylation of p38. Furthermore, the kinetics of E2F1-induced Wip1 expression highly correlated with the kinetics of E2F1-induced p38 dephosphorylation (Fig. 7A, lanes 1–4, compare upper and lower panels). Next, we tested whether induction of Wip1 plays an important role in E2F1-induced apoptosis. Activation of E2F1 in U2OS cells expressing ER-E2F1 resulted in apoptotic cell death as determined by the appearance of cells with sub-G1 DNA content. Importantly, this E2F1-induced apoptosis was significantly augmented by Wip1-specific siRNA (Fig. 8, A and B). An irrelevant siRNA did not effect Wip1 expression and E2F1-induced apoptosis (data not shown). Activation of E2F1 also led to an increase in the percentage of cells in S-phase; however, Wip1-specific siRNA did not significantly affect this E2F1-induced S-phase entry (Fig. 8A). These data indicate that Wip1, which is an E2F-regulated gene, negatively regulates E2F1-induced apoptosis, suggesting the existence of an E2F1-Wip1 negative feedback loop. E2F Induces p38 Phosphorylation—The pRB-E2F pathway is a downstream target of various mitogenic signaling cascades. We have recently demonstrated that E2F positively affects AKT activation (3Chaussepied M. Ginsberg D. Mol. Cell. 2004; 16: 831-837Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). We have shown here that E2F activation leads to a transient increase in phosphorylation of the MAPK p38, providing another example for the effect of E2F on signal transduction pathways. Thus, in addition to the well documented flow of information from surface receptors to E2F, there is a reverse input from E2F to upstream components of signaling pathways. The E2F-induced increase in p38 phosphorylation is achieved through the transcriptional induction of the kinase ASK1, a member of the MAPKKK family that phosphorylates p38 MKKs. We have shown that ASK1 is a transcriptional target of E2F1 and that siRNA directed against ASK1 inhibits E2F1-induced p38 phosphorylation. Knock down of ASK1 did not fully inhibit E2F1-induced p38 phosphorylation, suggesting that additional E2F-regulated genes may contribute to the p38 phosphorylation. Previously published data indicate that there are a few functional connections between E2F and the ASK1/p38 pathway. First, p38 can induce phosphorylation of pRB that is Cdk independent (29Wang S. Nath N. Minden A. Chellappan S. EMBO J. 1999; 18: 1559-1570Crossref PubMed Scopus (111) Google Scholar). This p38-induced phosphorylation of pRB correlates with a dissociation of E2F from pRb-E2F complexes and increased E2F-mediated transcription (29Wang S. Nath N. Minden A. Chellappan S. EMBO J. 1999; 18: 1559-1570Crossref PubMed Scopus (111) Google Scholar). In addition, in response to specific apoptotic stimuli ASK1 binds pRB and this is correlated with an increase in E2F transcriptional activity (30Dasgupta P. Betts V. Rastogi S. Joshi B. Morris M. Brennan B. Ordonez-Ercan D. Chellappan S. J. Biol. Chem. 2004; 279: 38762-38769Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Our data show that E2F1 up-regulates ASK1 expression leading to E2F-induced p38 phosphorylation. Taken together, these data suggest the existence of an E2F/ASK1/p38 positive feedback loop. On the other hand, during keratinocyte differentiation, E2F1 protein levels are reduced due to proteasomal degradation and this process requires p38 activity (31Ivanova I.A. D'Souza S.J. Dagnino L. Oncogene. 2006; 25: 430-437Crossref PubMed Scopus (23) Google Scholar). This suggests that under particular physiological settings there might be a negative feedback loop between p38 and E2F. Thus, the nature of the E2F-p38 connection may be tissue and context dependent. The biological outcome(s) of E2F-induced p38 phosphorylation are yet to be determined. However, one interesting possibility is that p38 constitutes a novel functional link between E2F1 and p53. In response to UV irradiation, p38 directly phosphorylates the tumor suppressor p53 on serine 33 and serine 46, thereby stimulating p53-mediated transcription and apoptosis (28Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (594) Google Scholar). Activation of E2F1 also leads to phosphorylation of p53 on serine 46, and this modification was shown to be important for E2F1-p53 cooperation in apoptosis (32Hershko T. Chaussepied M. Oren M. Ginsberg D. Cell Death Differ. 2005; 12: 377-383Crossref PubMed Scopus (92) Google Scholar). Our results imply that E2F1 can mediate phosphorylation of serine 46 on p53 via activation of p38 kinase. Interestingly, both E2F1-induced p38 phosphorylation and E2F1-induced p53 phosphorylation on serine 46 are transient (Figs. 1 and 5 and Ref. 32Hershko T. Chaussepied M. Oren M. Ginsberg D. Cell Death Differ. 2005; 12: 377-383Crossref PubMed Scopus (92) Google Scholar). Elucidation of the role of p38 in E2F1-induced activation of p53 awaits further studies. Wip1 Is a Novel E2F-regulated Gene—As mentioned above, E2F1 induces a transient phosphorylation of p38. The reduction in p38 phosphorylation, detected late after E2F1 activation, can occur via various mechanisms, some of which may be E2F independent. However, our data indicate that this decrease in p38 phosphorylation is achieved, at least in part, via E2F-induced up-regulation of Wip1, a serine/threonine phosphatase that dephosphorylates and inactivates p38. We show that Wip1 is a novel transcriptional target of E2F. Wip1 was shown to be a transcriptional target of p53, raising the possibility that E2F induces Wip1 expression via p53. However, our data demonstrate that E2F regulates Wip1 expression also in p53-deficient cells, indicating that E2F regulates Wip1 expression in a p53-independent manner. Importantly, inhibition of Wip1 expression, using specific siRNA, prolongs E2F1-induced p38 phosphorylation, indicating that Wip1 is an important mediator of E2F1-induced dephosphorylation of p38. Of note, when E2F1 induces Wip1 expression the levels of ASK1 are still high; nevertheless, Wip1 seems to reduce the phosphorylation of p38 even in the presence of ASK1. The Wip1 gene is a bona fide oncogene, and it is amplified and overexpressed in many human tumors (33Bulavin D.V. Demidov O.N. Saito S. Kauraniemi P. Phillips C. Amundson S.A. Ambrosino C. Sauter G. Nebreda A.R. Anderson C.W. Kallioniemi A. Fornace Jr., A.J. Appella E. Nat. Genet. 2002; 31: 210-215Crossref PubMed Scopus (360) Google Scholar, 34Li J. Yang Y. Peng Y. Austin R.J. van Eyndhoven W.G. Nguyen K.C. Gabriele T. McCurrach M.E. Marks J.R. Hoey T. Lowe S.W. Powers S. Nat. Genet. 2002; 31: 133-134Crossref PubMed Scopus (214) Google Scholar, 35Saito-Ohara F. Imoto I. Inoue J. Hosoi H. Nakagawara A. Sugimoto T. Inazawa J. Cancer Res. 2003; 63: 1876-1883PubMed Google Scholar, 36Hirasawa A. Saito-Ohara F. Inoue J. Aoki D. Susumu N. Yokoyama T. Nozawa S. Inazawa J. Imoto I. Clin. Cancer Res. 2003; 9: 1995-2004PubMed Google Scholar). Moreover, cells and animals deficient of Wip1 are resistant to transformation and oncogene-induced mammary tumors, emphasizing its pivotal role in tumorigenesis (37Bulavin D.V. Phillips C. Nannenga B. Timofeev O. Donehower L.A. Anderson C.W. Appella E. Fornace Jr., A.J. Nat. Genet. 2004; 36: 343-350Crossref PubMed Scopus (356) Google Scholar). To date, Wip1 known substrates are p38, p53, the DNA damage-induced kinase Chk1, and the DNA repair enzyme uracil DNA glycosylase 2 (19Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (357) Google Scholar, 38Lu X. Bocangel D. Nannenga B. Yamaguchi H. Appella E. Donehower L.A. Mol. Cell. 2004; 15: 621-634Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 39Lu X. Nannenga B. Donehower L.A. Genes Dev. 2005; 19: 1162-1174Crossref PubMed Scopus (315) Google Scholar). Wip1-mediated dephosphorylation of its substrates invariably results in their inactivation: The inhibition of p38 activity by Wip1 attenuates UV-induced phosphorylation of p53 at serines 33 and 46, resulting in suppression of p53-mediated transcription and apoptosis (19Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (357) Google Scholar). In addition, Wip1 dephosphorylates p53 at ATM-targeted phospho-serine 15 (39Lu X. Nannenga B. Donehower L.A. Genes Dev. 2005; 19: 1162-1174Crossref PubMed Scopus (315) Google Scholar). Of note, the substrates of Wip1 are often positively regulated by E2F. E2F1 stabilizes p53 and induces its phosphorylations on serines 15, 33, and 46 (22Berkovich E. Ginsberg D. Oncogene. 2003; 22: 161-167Crossref PubMed Scopus (100) Google Scholar, 32Hershko T. Chaussepied M. Oren M. Ginsberg D. Cell Death Differ. 2005; 12: 377-383Crossref PubMed Scopus (92) Google Scholar, 40Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (810) Google Scholar, 41Powers J.T. Hong S. Mayhew C.N. Rogers P.M. Knudsen E.S. Johnson D.G. Mol. Cancer Res. 2004; 2: 203-214PubMed Google Scholar, 42Rogoff H.A. Pickering M.T. Frame F.M. Debatis M.E. Sanchez Y. Jones S. Kowalik T.F. Mol. Cell. Biol. 2004; 24: 2968-2977Crossref PubMed Scopus (104) Google Scholar, 43Rogoff H.A. Pickering M.T. Debatis M.E. Jones S. Kowalik T.F. Mol. Cell. Biol. 2002; 22: 5308-5318Crossref PubMed Scopus (92) Google Scholar). E2F up-regulates the expression of the DNA repair gene uracil DNA glycosylase 2 (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (630) Google Scholar, 8Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (222) Google Scholar, 9Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Crossref PubMed Scopus (909) Google Scholar, 12Stanelle J. Stiewe T. Theseling C.C. Peter M. Putzer B.M. Nucleic Acids Res. 2002; 30: 1859-1867Crossref PubMed Scopus (146) Google Scholar, 44Haug T. Skorpen F. Aas P.A. Malm V. Skjelbred C. Krokan H.E. Nucleic Acids Res. 1998; 26: 1449-1457Crossref PubMed Scopus (92) Google Scholar) and the damage-induced kinase Chk1 (45Carrassa L. Broggini M. Vikhanskaya F. Damia G. Cell Cycle. 2003; 2: 604-609Crossref PubMed Scopus (32) Google Scholar), and as shown here E2F1 induces p38 phosphorylation. Thus, the emerging picture suggests that Wip1 is an E2F-regulated gene that functions in inactivating E2F-induced proteins such as p38, p53, Chk1, and uracil DNA glycosylase 2. As novel substrates of Wip1 are identified, it will be interesting to test whether this pattern is valid for them too. Interestingly, we demonstrate that inhibition of Wip1 expression, using specific siRNA, enhances E2F1-induced apoptosis. This indicates that Wip1, which is an E2F-regulated gene, inhibits E2F1 activity, suggesting that E2F1 and Wip1 are components of a negative feedback loop. In summary, we have shown that E2F regulates the expression of a kinase, ASK1, and a phosphatase, Wip1, that modulate p38 activity. Consequently, E2F induces a transient activation of p38. This constitutes a novel link between the pRB/E2F pathway and a major signal transduction pathway. We thank Marie Chaussepied and Moshe Oren for helpful discussions, Yocheved Lamed for excellent technical assistance, and Haim Y. Cohen and Neri Pilosof for help with ChIP analysis. Anti-p38 antibodies were a generous gift from Prof. Roni Zeger, Weizmann Institute, Israel.