Title: Hepatitis B Virus X Protein via the p38MAPK Pathway Induces E2F1 Release and ATR Kinase Activation Mediating p53 Apoptosis
Abstract: Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocellular carcinoma (HCC) pathogenesis by an unknown mechanism. Deletions or mutations of genes involved in the p53 pathway are often associated with HBV-mediated HCC, indicating rescue from p53 apoptosis is a likely mechanism in HBV-HCC pathogenesis. Herein, we determined the mechanism by which pX sensitizes hepatocytes to p53-mediated apoptosis. Although it is well established that the Rb/E2F/ARF pathway stabilizes p53, and the DNA damage-activated ATM/ATR kinases activate p53, the mechanism that coordinates these two pathways has not been determined. We demonstrate that the p38MAPK pathway activated by pX serves this role in p53 apoptosis. Specifically, the activated p38MAPK pathway stabilizes p53 via E2F1-mediated ARF expression, and also activates the transcriptional function of p53 by activating ATR. Knockdown of p53, E2F1, ATR, or p38MAPKα abrogates pX-mediated apoptosis, demonstrating that E2F1, ATR, and p38MAPKα are all essential in p53 apoptosis in response to pX. Specifically, in response to pX expression, the p38MAPK pathway activates Cdk4 and Cdk2, leading to phosphorylation of Rb, release of E2F1, and transcription of ARF. The p38MAPK pathway also activates ATR, leading to phosphorylation of p53 on Ser-18 and Ser-23, transcription of pro-apoptotic genes Bax, Fas, and Noxa, and apoptosis. In conclusion, pX sensitizes hepatocytes to p53 apoptosis via activation of the p38MAPK pathway, which couples p53 stabilization and p53 activation, by E2F1 induction and ATR activation, respectively. Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocellular carcinoma (HCC) pathogenesis by an unknown mechanism. Deletions or mutations of genes involved in the p53 pathway are often associated with HBV-mediated HCC, indicating rescue from p53 apoptosis is a likely mechanism in HBV-HCC pathogenesis. Herein, we determined the mechanism by which pX sensitizes hepatocytes to p53-mediated apoptosis. Although it is well established that the Rb/E2F/ARF pathway stabilizes p53, and the DNA damage-activated ATM/ATR kinases activate p53, the mechanism that coordinates these two pathways has not been determined. We demonstrate that the p38MAPK pathway activated by pX serves this role in p53 apoptosis. Specifically, the activated p38MAPK pathway stabilizes p53 via E2F1-mediated ARF expression, and also activates the transcriptional function of p53 by activating ATR. Knockdown of p53, E2F1, ATR, or p38MAPKα abrogates pX-mediated apoptosis, demonstrating that E2F1, ATR, and p38MAPKα are all essential in p53 apoptosis in response to pX. Specifically, in response to pX expression, the p38MAPK pathway activates Cdk4 and Cdk2, leading to phosphorylation of Rb, release of E2F1, and transcription of ARF. The p38MAPK pathway also activates ATR, leading to phosphorylation of p53 on Ser-18 and Ser-23, transcription of pro-apoptotic genes Bax, Fas, and Noxa, and apoptosis. In conclusion, pX sensitizes hepatocytes to p53 apoptosis via activation of the p38MAPK pathway, which couples p53 stabilization and p53 activation, by E2F1 induction and ATR activation, respectively. p53 protects the integrity of the genome in response to stress signals including hypoxia, metabolic stress, DNA damage, and expression of cellular and viral oncoproteins by inducing growth arrest, senescence, or apoptosis (1Kinzler K.W. Vogelstein B. Nature. 1997; 386: 761-763Crossref PubMed Scopus (996) Google Scholar, 2Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1167) Google Scholar, 3Lowe S.W. Ruley H.T. Genes Dev. 1993; 7: 535-545Crossref PubMed Scopus (609) Google Scholar). p53 stabilization by overexpression of cellular or viral oncoproteins is well established, involving E2F1-mediated ARF expression (3Lowe S.W. Ruley H.T. Genes Dev. 1993; 7: 535-545Crossref PubMed Scopus (609) Google Scholar, 4de Stanchina E. McCurrach M.E. Zindy F. Shieh S.-Y. Ferbeyer G. Samuelson A.V. Prives C. Roussel M.F. Sherr C.J. Lowe S. Genes Dev. 1998; 12: 2434-2442Crossref PubMed Scopus (544) Google Scholar, 5Ginsberg D. FEBS Lett. 2002; 529: 122-125Crossref PubMed Scopus (152) Google Scholar). Activated p53 exhibits increased half-life after dissociation from Mdm2 (6Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar). This p53 stabilization step involves Rb phosphorylation, E2F1 release, and induction of Arf gene transcription (7Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (809) Google Scholar). ARF sequesters Mdm2 from interaction with p53, decreasing p53 degradation thereby enhancing p53 stability (8Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1321) Google Scholar). In response to stress, p53 becomes heavily phosphorylated (2Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1167) Google Scholar), inhibiting Mdm2 binding and p53 degradation (6Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar). Transcriptional activation of p53 involves multisite modifications, including phosphorylation and acetylation (2Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1167) Google Scholar, 9Tang Y. Luo J. Zhang W. Gu W. Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). ATM and ATR kinases phosphorylate Ser-15 and Ser-20 of p53 following DNA damage (10Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1684) Google Scholar, 11Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell Biol. 2000; 1: 179-186Crossref PubMed Scopus (652) Google Scholar). However, despite the plethora of studies dealing with p53 stabilization and p53 transcriptional activation, how these two pathways become simultaneously activated to affect p53 apoptosis remains to be determined. Deletions or mutations of p53 (12Hainaut P. Hernandez T. Robinson A. Rodriguez-Tome P. Flores T. Hollstein M. Harris C.C. Montesano R. Nucleic Acids Res. 1998; 26: 205-213Crossref PubMed Scopus (411) Google Scholar) as well as disruption of p53 activation mechanisms, such as loss of Rb, Arf, or amplification of genes that inactivate the cellular stress pathways (13Bulavin 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 (353) Google Scholar) are linked to cancer (14Greenblatt M.S. Bennett W.P. Hollstein M. Harris C.C. Cancer Res. 1994; 54: 4855-4878PubMed Google Scholar), including Hepatitis B virus (HBV) 2The abbreviations used are: HBVhepatitis B virusHCChepatocellular carcinomaMAPKmitogen-activated protein kinaseGSTglutathione S-transferaseChIPchromatin immunoprecipitation assayJNKc-Jun N-terminal kinaseWCEwhole cell extractGAPDHglyceraldehyde-3-phosphate dehydrogenasekdknockdownRbretinoblastoma protein. 2The abbreviations used are: HBVhepatitis B virusHCChepatocellular carcinomaMAPKmitogen-activated protein kinaseGSTglutathione S-transferaseChIPchromatin immunoprecipitation assayJNKc-Jun N-terminal kinaseWCEwhole cell extractGAPDHglyceraldehyde-3-phosphate dehydrogenasekdknockdownRbretinoblastoma protein.-mediated hepatocellular carcinoma (HCC) (15Buendia M.A. Semin Cancer Biol. 2000; 10: 185-200Crossref PubMed Scopus (282) Google Scholar). 50% of HCCs are caused by chronic HBV infection (16Parkin D.M. Bray F.I. Devesa S.S. Eur. J. Cancer. 2001; 37: S4-66Abstract Full Text Full Text PDF PubMed Google Scholar). The World Health Organization reports 400 million people are chronically infected with HBV, placing them at a greatly increased risk for HCC development. The 16.5-kDa X protein (pX) encoded by HBV is required for the viral life cycle and is implicated in HCC pathogenesis (17Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). pX is multifunctional, inducing activation of the mitogenic Ras-Raf-MAPK, JNK, and p38MAPK pathways (18Bouchard M.J. Schneider R.J. J. Virol. 2004; 78: 12725-12734Crossref PubMed Scopus (402) Google Scholar), and transcription of select viral and cellular genes (17Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). The consequence of these pX activities is deregulation of cellular gene expression resulting in unscheduled cell cycle progression (19Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11215-11219Crossref PubMed Scopus (264) Google Scholar, 20Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Kim Y. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (117) Google Scholar), apoptosis (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar), and transformation of differentiated hepatocytes (22Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Genes transcriptionally induced by pX and linked to deregulated cell growth and apoptosis include the cyclins (23Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), TNFα/TNFRI, Fas/FasL, and p53-responsive genes (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Importantly, expression of pX is preferentially maintained in HCC (24Su Q. Schroder C.H. Hofmann W.J. Otto G. Pichlmayr R. Bannasch P. Hepatology. 1998; 27: 1109-1120Crossref PubMed Scopus (191) Google Scholar), although the mechanism by which pX contributes to HCC development remains to be determined. HBV-mediated HCC often exhibit genetic alterations in the tumor suppressor genes p53, Rb, and Arf (15Buendia M.A. Semin Cancer Biol. 2000; 10: 185-200Crossref PubMed Scopus (282) Google Scholar), indicating that rescue from p53 apoptosis is a likely mechanism of HBV-mediated HCC (25Thorgeirsson S.S. Grisham J.W. Nat. Genet. 2002; 31: 339-346Crossref PubMed Scopus (1256) Google Scholar). However, the mechanism by which pX activates p53 has not been determined. hepatitis B virus hepatocellular carcinoma mitogen-activated protein kinase glutathione S-transferase chromatin immunoprecipitation assay c-Jun N-terminal kinase whole cell extract glyceraldehyde-3-phosphate dehydrogenase knockdown retinoblastoma protein. hepatitis B virus hepatocellular carcinoma mitogen-activated protein kinase glutathione S-transferase chromatin immunoprecipitation assay c-Jun N-terminal kinase whole cell extract glyceraldehyde-3-phosphate dehydrogenase knockdown retinoblastoma protein. In this study, we investigate the mechanism of p53 activation by pX, employing the well-characterized 4pX-1 cell line (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar, 22Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 23Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar); 4pX-1 cells conditionally express pX via the tet-off system. The significance of this cellular model is that pX expression is very low, resembling expression levels occurring in natural HBV infection (22Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In our earlier studies we have shown that pX sensitizes 4pX-1 cells to apoptosis only when challenged by additional sub-apoptotic signals such as growth factor deprivation, by activating the p38MAPK pathway (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Importantly, we have shown that pX expression induces sustained activation of the p38MAPK pathway within 6 h following serum withdrawal (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). By contrast, in the absence of pX expression, serum withdrawal induces the p38MAPK pathway later, at 24 h. Thus, the effect of pX on the activation of the p38MAPK pathway precedes the effect of serum withdrawal. Moreover, inhibition of the p38MAPK pathway by SB 202190 inhibits pX-mediated apoptosis. Accordingly, our earlier studies (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar) have clearly established that the activation of the p38MAPK pathway by pX is necessary for initiation of pX-mediated apoptosis. The role of the p38MAPK pathway in pX-mediated apoptosis remains to be determined. Herein we demonstrate that the p38MAPK pathway, activated by pX following growth factor withdrawal, induces p53 stabilization by E2F1-mediated ARF expression. The p38MAPK pathway also induces the transcriptional activity of p53 by activating ATR, leading to phosphorylation of Ser-18 and Ser-23 of murine p53. Knockdown of p53, E2F1, ATR, or p38MAPKα abrogates pX-mediated apoptosis, identifying the essential role of the p38MAPK pathway, E2F1, and ATR in p53 apoptosis. We conclude that activation of the p38MAPK pathway by the weakly oncogenic pX couples the stabilization and activation of p53, via ARF induction and ATR activation, respectively, leading to p53-mediated apoptosis. Cell Culture—The 4pX-1 cell line, derived from AML12 cells, was propagated as described (22Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) in medium containing 5 μg/ml tetracycline to turn-off pX expression. Apoptotic conditions are as described (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Briefly, confluent 4pX-1 cells were treated ± 5 μg/ml tetracycline for 24 h in medium containing 10% fetal calf serum; apoptosis was initiated by switching cultures to 2% fetal calf serum ± 5 μg/ml tetracycline. Concentrations of inhibitors were: SB202190 (CalBiochem), 5 μm; SP600125 (Tocris), 5 μm; PFT-α (BioMoL), 10 μm; caffeine, 8 mm; cycloheximide, 10 μm. Transient Transfections—Transient transfections of luciferase reporters were performed using FuGENE 6 (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). DNA was added to apoptotic 4pX-1 cultures 8 h prior to serum withdrawal; cells were harvested after 24 h. Assays were performed in triplicate and quantified per μg of protein extract. NFκB-Luc, NFAT-Luc, p53-Luc were purchased from Stratagene. Fas (-1.7 kb)-Luc was kindly provided by Dr. Owens-Schaub. Construction of Clonal 4pX-1-p53kd, 4pX-1-E2F1kd, 4pX-1-ATRkd, and p38MAPKkd Cell Lines—4pX-1 cells were transfected with pSilencer CMV-Puro (Ambion) containing the p53 sequence forming a short hairpin (26Dirac A.M. Bernards R. J. Biol. Chem. 2003; 278: 11731-11734Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), or the E2F1 and ATR sequences listed below. The p38MAPKα-shRNA vector was purchased from Open Biosystems. Clonal stable cell lines were isolated by puromycin (1.0 μg/ml) selection and screened by Western blot assays employing antibodies for p53, E2F1, ATR and p38MAPKα. Clonal stable cell lines exhibiting the highest level of knockdown were selected for further analyses. Clonal stable cell lines isolated from the same cultures exhibiting absence of knockdown referred to as pseudo-knockdown cell lines (pseudokd), serve as the negative control: E2F1shRNA: 5′-GATCCACGGAGGCTGGATCTGGAGTTCAAGAGACTCCAGATCCAGCCTCCGTTTTTTTGGAAT-3′; ATRshRNA: 5′-GATCCTCTACATCATCTTTGTAAGTTCAAGAGACTTACAAAGATGATGTAGATTT-3′. Real-time Quantitative PCR—Real-time quantitative PCR was performed as described (23Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), employing 18 S RNA as internal control. Primers used were: ARF: Fwd, 5′-ATAGCTTCAGCTCAAGCACG-3′ and Rev, 5′-AAGCCACATGCTAGACACG-3′; ASPP2: Fwd, 5′-GGAGATCGAGCAGATGAATAGC-3′ and Rev, 5′-ATCCTGCCGTTCTTCAGC-3′; CHK2: Fwd, 5′-AACCTGAAGAACCTGGTCC-3′ and Rev, 5′-TCGAAGCAATATTCACAGC-3′; pX: Fwd, 5′-TCCCAGCAATGTCAACGACC-3′ and Rev, 5′-CCAATTTATGCCTACAGCCTCC-3′. DNA fragmentation assays were performed as described (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). In vitro Cdk4 kinase assays were performed as described (23Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Western Blot Analyses—Western blot analyses were performed employing whole cell extracts (WCE) isolated from apoptotic cultures (107 cells), grown ±5 μg/ml tetracycline, and harvested in 200 μl of 1× SDS loading buffer. Western blot analyses were performed using Amersham Biosciences ECL reagent. Antibodies used were purchased from: p53 (Vector); phospho-Ser18-p53, phospho-Ser23-p53, and phospho-Ser389-p53, Rb, phospho-Ser800/804-Rb, active caspase3, phospho-ATR, Cdk2, and phospho-Y15-Cdc2 (Cell Signaling); ATM, phospho-Ser1987-ATM (Calbiochem); ATR (Abcam); E2F1 (Activemotif); ARF (Novus). Chromatin Immunoprecipitation (ChIP) Assays—ChIP was performed as described (27Benjanirut C. Paris M. Wang W.-H. Hong S. Kim K. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2006; 281: 2969-2981Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 28Paris M. Wang W.-H. Shin M.-H. Franklin D.S. Andrisani O.M. Mol. Cell Biol. 2006; 26: 8826-8839Crossref PubMed Scopus (26) Google Scholar) employing 3 μg of ChIP-validated E2F1 (Activemotif) antibody. Immunoprecipitated DNA was quantified by real-time PCR or following PCR amplification by agarose gel electrophoresis. Sequences of the forward and reverse primers flanking the functional E2F1 binding sites of the Arf, Chk2, and ASPP2 promoters are: ARF: Fwd, 5′-GCGGCGCTGGCTGTCACCGCG-3′ and Rev, 5′-CCTCAGCGGCGGCCTCACCG-3′; ASPP2: Fwd, 5′-ACATCAGGCATGTATGACATT-3′ and Rev, 5′-CTGCTGAACTGGAAACCCCA-3′; Chk2: Fwd, 5′-TAGGTAGCAAGACCCGAGGG-3′ and Rev, 5′-CCTTCCTCCCCGGCAGGACC-3′. pX expression Induces p53-mediated Apoptosis—The HBV X protein sensitizes the 4pX-1 hepatocyte cell line to apoptosis after growth factor withdrawal by inducing sustained activation of the p38MAPK pathway (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Specifically, 4pX-1 cultures expressing pX via the Tet-off expression system (22Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), exhibit sustained activation of the p38MAPK pathway within 6 h following growth factor withdrawal. By contrast, in the absence of pX expression, serum withdrawal induces the p38MAPK pathway later at 24 h. Thus, the effect of pX on the activation of the p38MAPK pathway precedes the effect of serum withdrawal. Moreover, inhibition of the p38MAPK pathway by SB 202190 inhibits pX-mediated apoptosis. Thus, our earlier studies (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar) established that the activation of the p38MAPK pathway by pX is necessary for initiation of pX-mediated apoptosis. Moreover, the pX-mediated activation of the p38MAPK pathway induces expression of TNFRI/TNF-α, Fas/FasL, and p53-regulated genes Fas, Bax, and Noxa (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Expression of pro-apoptotic p53-regulated genes in response to pX suggested the involvement of p53 in pX-mediated apoptosis, in agreement with earlier observations (30Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (192) Google Scholar). The mechanism by which pX stabilizes and activates pro-apoptotic p53 transcription is unknown. To obtain initial indications whether pX activates p53 transcription, the p53-responsive luciferase constructs p53-Luc, pBax-Luc (23Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and pFas-Luc (31Cerrato J.A. Khan T. Koul D. Lang F.F. Conrad C.A. Yung W.K. Liu T.J. Int. J. Oncol. 2004; 24: 409-417PubMed Google Scholar) were transiently transfected in apoptotic 4pX-1 cells. All p53-responsive promoters display induction in response to pX expression (Fig. 1A). Treatment with 10 μm PFT-α, known to specifically inhibit p53 transcriptional activity (29Murphy P.J. Galigniana M.D. Morishima Y. Harrell J.M. Kwok R.P. Ljungman M. Pratt W.B. J. Biol. Chem. 2004; 279: 30195-30201Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), inhibits induction by pX from the p53-dependent reporter, without significantly inhibiting NFκB- or NFAT-driven luciferase expression, suggesting pX activates p53 (Fig. 1A). To confirm the effect of pX on p53 transcriptional activation and pX-mediated apoptosis, we examined expression of the endogenous p53-regulated genes Bax and p21, in a time course, following incubation of 4pX-1 cells in apoptotic conditions by growth factor withdrawal (Fig. 1B). pX induces expression of both p21 and Bax, as early as 3 h following incubation in apoptotic conditions. Importantly, by 12 h pX mediates a 3-fold increase in the expression of pro-apoptotic Bax in comparison to a 1.6-fold increase in expression of the growth arrest gene p21, suggesting the involvement of p53 in apoptosis, in response to pX expression. To determine whether p53 activation is involved in pX-dependent apoptosis, radioactive DNA fragmentation assays were performed in apoptotic 4pX-1 cultures, as a function of PFT-α addition (Fig. 1C). PFT-α profoundly inhibits onset of apoptosis in response to pX (Fig. 1C). Likewise, PFT-α inhibits pX-dependent caspase3 activation and apoptosis, assayed by the cell-permeable fluorigenic Z-DEVD-FMK caspase3 substrate (Fig. 1D). To directly demonstrate involvement of p53 in pX-mediated apoptosis, a 4pX-1 p53 knockdown (kd) cell line was generated, 4pX-1-p53kd, displaying more than 90% silencing of p53. p53-responsive luciferase plasmids transfected in the 4pX-1-p53kd cell line do not exhibit pX-dependent induction. Importantly, this cell line is resistant to apoptosis in response to pX expression (Fig. 1D), assayed by immunofluorescence microscopy, using the cell-permeable fluorigenic Z-DEVD-FMK caspase3 substrate (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Together, these results (Fig. 1) support that pX activates p53 and induces p53-mediated apoptosis. p53 Stabilization by pX Requires Activation of the p38MAPK Pathway—Our earlier studies (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar) have linked apoptosis by pX to activation of the p38MAPK pathway. Because our results now show that p53 mediates apoptosis in response to pX expression (Fig. 1), we investigated the link between the p38MAPK pathway and p53. A p38MAPKα knockdown cell line (4pX-1-p38MAPKαkd) was constructed, exhibiting ∼80% reduction in the endogenous p38MAPKα (Fig. 2A). Employing the 4pX-1 and 4pX-1-p38MAPKαkd cell lines, we investigated whether pX stabilizes p53, and if p53 stability is dependent on activation of the p38MAPK pathway. Confluent, 4pX-1, and 4pX-1-p38MAPKαkd cultures were grown as a function of pX expression, and apoptosis was initiated by serum withdrawal (21Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar). Cycloheximide was added 2 h after onset of apoptosis. p53 protein levels were determined by Western blot analyses and quantified in a time course, following cycloheximide addition (Fig. 2, B and C). In 4pX-1 cells grown in the absence of pX, 50% of p53 remains 30 min after cycloheximide addition, reaching basal levels by 120 min. By contrast, in the presence of pX, 80% of p53 remains at the 30-min interval, decreasing to 60% by 60 min, and reaching the basal level 180 min after cycloheximide addition. The half-life of p53 is estimated to be ∼30 min in apoptotic 4pX-1 cultures without pX, whereas in the presence of pX, the p53 half-life is increased to 75 min, demonstrating that pX stabilizes p53. Interestingly, in 4pX-1-p38MAPKαkd cells expressing pX, knockdown of the p38MAPKα decreased the half-life of p53 to 45 min, indicating the p38MAPK pathway plays a role in pX-mediated stabilization of p53. Similar conclusions were derived from pulse-chase studies of p53, employing in vivo metabolic labeling with [35S]methionine, in conjunction with treatment using the p38MAPK pathway inhibitor SB202190 (data not shown). The p38MAPK Pathway Activated by pX Promotes E2F1 Function via Cdk4/Cdk2 Activation—The E2F family of transcription factors is sequestered from function by binding to unphosphorylated Rb. Activated Cdk4 and Cdk2 phosphorylate Rb, releasing E2Fs which mediate transcription of either proliferative S-phase genes, or pro-apoptotic genes such as Arf (7Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (809) Google Scholar, 32Moon N.S. Frolov M.V. Kwon E.J. De Stefano L. Dimova D.K. Morris E.J. Taylor-Harding B. White K. Dyson N.J. Dev. Cell. 2005; 9: 463-475Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To obtain initial indications whether pX induces E2F1 activity, the E2F1-responsive APAF-Luc (33Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (226) Google Scholar) and CyclA-Luc (34Wells J. Graveel C.R. Bartley S.M. Madore S.J. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3890-3895Crossref PubMed Scopus (122) Google Scholar) reporters were transiently transfected in apoptotic 4pX-1 cultures. pX expression promoted a 3–4-fold luciferase induction (Fig. 3A), suggesting that pX mediates E2F1 release. Based on these observations, we determined the phosphorylation status of Rb in pX-expressing cells grown in apoptotic conditions. Enhanced Rb phosphorylation was observed in apoptotic 4pX-1 cultures expressing pX, detected by Western blot analyses with the phospho-Rb Ser-800/804 antibody (Fig. 3B). Specifically, in pX-expressing 4pX-1 cells, phosphorylation of Rb increased by 8-fold starting 0 to 12 h after growth factor withdrawal. By contrast, in 4pX-1-p38MAPKαkd cells expressing pX, only a 1.5-fold increase in Rb phosphorylation is observed (Fig. 3B), supporting that the pX-activated p38MAPK pathway is required for Rb phosphorylation. Because Rb phosphorylation is mediated by G1 phase cyclin-dependent kinases, we examined by in vitro immunocomplex kinase assays, the level of Cdk4 activation as a function of pX expression (Fig. 3C). Employing extracts isolated from apoptotic 4pX-1 cells, we demonstrate that Cdk4 activation is maintained for 12 h following onset of apoptosis in the presence of pX. Inhibition of the p38MAPK pathway by SB202190 suppresses this pX-dependent Cdk4 activation, whereas inhibition of the JNK pathway by treatment with SP600125 has only a small effect (Fig. 3C). To further confirm these results, we also examined the activation state of Cdk2, and its link to the p38MAPK pathway. A critical step in Cdk2 activation, similar to Cdc2 activation (35Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2568) Google Scholar), is dephosphorylation of Tyr-15 (36Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2916) Google Scholar). Employing extracts from the 4pX-1 and 4pX-1-p38MAPKαkd cell lines, we monitored by Western blot analyses the phosphorylation state of Tyr-15 of Cdk2 as a function of pX expression (Fig. 3D). In 4pX-1 cells grown in apoptotic conditions for 12 h, pX expression results in Tyr-15 dephosphorylation of Cdk2, indicating pX mediates activation of Cdk2. By contrast, the inhibitory Tyr-15 phosphorylation is maintained in 4pX-1 cells not expressing pX, as well as in pX-expressing cells in which p38MAPKα has been knocked-down (4pX-1-p38MAPKαkd cells). We interpret these results to mean that pX cannot activate Cdk2 in the absence of the p38MAPK pathway. We conclude activation of the p38MAPK by pX expression mediates activation of Cdk4 and Cdk2, in turn phosphorylating Rb. Phosphorylation of Rb results in release of E2F leading to transcription of E2F-responsive genes. Functional E2F1 binding sites have been mapped in the promoters of Arf (7Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K