JNK1-dependent PUMA Expression Contributes to Hepatocyte Lipoapoptosis
2009; Elsevier BV; Volume: 284; Issue: 39 Linguagem: Inglês
10.1074/jbc.m109.022491
ISSN1083-351X
AutoresSophie C. Cazanave, Justin L. Mott, Nafisa A. Elmi, Steven F. Bronk, Nathan W. Werneburg, Yuko Akazawa, Alişan Kahraman, Sean Garrison, Gerard P. Zambetti, Michael Charlton, Gregory J. Gores,
Tópico(s)Lipid metabolism and biosynthesis
ResumoFree fatty acids (FFA) induce hepatocyte lipoapoptosis by a c-Jun N-terminal kinase (JNK)-dependent mechanism. However, the cellular processes by which JNK engages the core apoptotic machinery during lipotoxicity, especially activation of BH3-only proteins, remain incompletely understood. Thus, our aim was to determine whether JNK mediates induction of BH3-only proteins during hepatocyte lipoapoptosis. The saturated FFA palmitate, but not the monounsaturated FFA oleate, induces an increase in PUMA mRNA and protein levels. Palmitate induction of PUMA was JNK1-dependent in primary murine hepatocytes. Palmitate-mediated PUMA expression was inhibited by a dominant negative c-Jun, and direct binding of a phosphorylated c-Jun containing the activator protein 1 complex to the PUMA promoter was identified by electrophoretic mobility shift assay and a chromatin immunoprecipitation assay. Short hairpin RNA-targeted knockdown of PUMA attenuated Bax activation, caspase 3/7 activity, and cell death. Similarly, the genetic deficiency of Puma rendered murine hepatocytes resistant to lipoapoptosis. PUMA expression was also increased in liver biopsy specimens from patients with non-alcoholic steatohepatitis as compared with patients with simple steatosis or controls. Collectively, the data implicate JNK1-dependent PUMA expression as a mechanism contributing to hepatocyte lipoapoptosis. Free fatty acids (FFA) induce hepatocyte lipoapoptosis by a c-Jun N-terminal kinase (JNK)-dependent mechanism. However, the cellular processes by which JNK engages the core apoptotic machinery during lipotoxicity, especially activation of BH3-only proteins, remain incompletely understood. Thus, our aim was to determine whether JNK mediates induction of BH3-only proteins during hepatocyte lipoapoptosis. The saturated FFA palmitate, but not the monounsaturated FFA oleate, induces an increase in PUMA mRNA and protein levels. Palmitate induction of PUMA was JNK1-dependent in primary murine hepatocytes. Palmitate-mediated PUMA expression was inhibited by a dominant negative c-Jun, and direct binding of a phosphorylated c-Jun containing the activator protein 1 complex to the PUMA promoter was identified by electrophoretic mobility shift assay and a chromatin immunoprecipitation assay. Short hairpin RNA-targeted knockdown of PUMA attenuated Bax activation, caspase 3/7 activity, and cell death. Similarly, the genetic deficiency of Puma rendered murine hepatocytes resistant to lipoapoptosis. PUMA expression was also increased in liver biopsy specimens from patients with non-alcoholic steatohepatitis as compared with patients with simple steatosis or controls. Collectively, the data implicate JNK1-dependent PUMA expression as a mechanism contributing to hepatocyte lipoapoptosis. Non-alcoholic fatty liver disease is commonly associated with the metabolic syndrome and affects up to one-third of the American population (1Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2876) Google Scholar). 5–10% of non-alcoholic fatty liver disease patients develop hepatic inflammation, a syndrome referred to as non-alcoholic steatohepatitis (NASH) 2The abbreviations used are: NASHnon-alcoholic steatohepatitisFFAfree fatty acid(s)AP-1activator protein-1BH3Bcl-2 homology domainEMSAelectrophoretic mobility shift assayJNKc-Jun-N-terminal kinaseMAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKMAPK kinase kinasePUMAp53-up-regulated mediator of apoptosisWTwild typeMLK3mixed lineage kinase 3DNdominant negativePBSphosphate-buffered salinePIPES1,4-piperazinediethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 2The abbreviations used are: NASHnon-alcoholic steatohepatitisFFAfree fatty acid(s)AP-1activator protein-1BH3Bcl-2 homology domainEMSAelectrophoretic mobility shift assayJNKc-Jun-N-terminal kinaseMAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKMAPK kinase kinasePUMAp53-up-regulated mediator of apoptosisWTwild typeMLK3mixed lineage kinase 3DNdominant negativePBSphosphate-buffered salinePIPES1,4-piperazinediethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 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JNK activation can be further self-amplified via a feed forward phosphorylation and activation of MLK3 by JNK (21Xu Z. Kukekov N.V. Greene L.A. Mol. Cell. Biol. 2005; 25: 9949-9959Crossref PubMed Scopus (51) Google Scholar, 22Schachter K.A. Du Y. Lin A. Gallo K.A. J. Biol. Chem. 2006; 281: 19134-19144Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar).Both JNK1 and -2 have been implicated in liver injury, although JNK1 is more strongly associated with steatohepatitis (17Singh R. Wang Y. Xiang Y. Tanaka K.E. Gaarde W.A. Czaja M.J. Hepatology. 2009; 49: 87-96Crossref PubMed Scopus (175) Google Scholar, 18Schattenberg J.M. Singh R. Wang Y. Lefkowitch J.H. Rigoli R.M. Scherer P.E. Czaja M.J. Hepatology. 2006; 43: 163-172Crossref PubMed Scopus (315) Google Scholar). JNK can cause cell death signals by both transcriptional and post-transcriptional mechanisms (23Czaja M.J. Am. J. Physiol. Gastrointest. 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This group of proteins includes Bad, Bid, Bik, Bim, Bmf, Hrk, NOXA, and PUMA, which display sequence conservation exclusively in the short (9–16 amino acids) BH3 (Bcl-2 homology 3) region, which is necessary for their ability to induce apoptosis. BH3-only proteins such as Bid, Bim, and PUMA can directly activate the multidomain pro-apoptotic members of the Bcl-2 family Bax and Bak (31Kim H. Rafiuddin-Shah M. Tu H.C. Jeffers J.R. Zambetti G.P. Hsieh J.J. Cheng E.H. Nat. Cell Biol. 2006; 8: 1348-1358Crossref PubMed Scopus (695) Google Scholar, 32Ming L. Wang P. Bank A. Yu J. Zhang L. J. Biol. Chem. 2006; 281: 16034-16042Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 33Neise D. Graupner V. Gillissen B.F. Daniel P.T. Schulze-Osthoff K. Jänicke R.U. Essmann F. Oncogene. 2008; 27: 1387-1396Crossref PubMed Scopus (27) Google Scholar, 34Gallenne T. Gautier F. Oliver L. Hervouet E. Noël B. Hickman J.A. Geneste O. Cartron P.F. Vallette F.M. Manon S. Juin P. J. Cell Biol. 2009; 185: 279-290Crossref PubMed Scopus (128) Google Scholar). The oligomerization of Bax and Bak in the outer mitochondrial membrane results in mitochondrial dysfunction, downstream activation of the effector caspases 3, 6, and 7, and ultimately cell death by apoptosis (35Taylor R.C. Cullen S.P. Martin S.J. Nat. Rev. Mol. Cell Biol. 2008; 9: 231-241Crossref PubMed Scopus (1871) Google Scholar). Among the BH3-only proteins, Bim has been identified as contributing to lipoapoptosis (12Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Indeed, saturated FFA up-regulates Bim expression, and small interfering RNA targeted knockdown of Bim partially attenuates FFA-mediated apoptosis (12Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Because the reduction in apoptosis was incomplete, it appears that in addition to Bim, other BH3-only proteins with complementary functions contribute to lipoapoptosis.PUMA (p53 up-regulated modulator of apoptosis) is a potent pro-apoptotic protein whose cellular levels are transcriptionally regulated by both p53-dependent and -independent mechanisms (36You H. Pellegrini M. Tsuchihara K. Yamamoto K. Hacker G. Erlacher M. Villunger A. Mak T.W. J. Exp. Med. 2006; 203: 1657-1663Crossref PubMed Scopus (335) Google Scholar, 37Hershko T. Ginsberg D. J. Biol. Chem. 2004; 279: 8627-8634Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 38Melino G. Bernassola F. Ranalli M. Yee K. Zong W.X. Corazzari M. Knight R.A. Green D.R. Thompson C. Vousden K.H. J. Biol. Chem. 2004; 279: 8076-8083Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 39Jeffers J.R. Parganas E. 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Med. 2006; 203: 2939-2951Crossref PubMed Scopus (189) Google Scholar). Two BH3-domain-containing isoforms of PUMA have been identified in humans, an α isoform (23 kDa) and a β isoform (18 kDa), both capable of efficiently inducing apoptosis (41Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar). Although PUMA has been extensively studied in several organs including colon cancer cells, thymocytes, and neurons (32Ming L. Wang P. Bank A. Yu J. Zhang L. J. Biol. Chem. 2006; 281: 16034-16042Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 40Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 44Steckley D. Karajgikar M. Dale L.B. Fuerth B. Swan P. Drummond-Main C. Poulter M.O. Ferguson S.S. Strasser A. Cregan S.P. J. Neurosci. 2007; 27: 12989-12999Crossref PubMed Scopus (120) Google Scholar, 45Villunger A. Michalak E.M. Coultas L. Müllauer F. Böck G. Ausserlechner M.J. Adams J.M. Strasser A. Science. 2003; 302: 1036-1038Crossref PubMed Scopus (1081) Google Scholar), its contribution to liver injury remains unexplored.The findings of this study suggest that PUMA contributes to FFA-induced lipoapoptosis in liver cells. The saturated FFA palmitate induces PUMA expression by a JNK1/AP-1 signaling cascade. PUMA up-regulation is also demonstrated in human liver samples from patients with NASH, strengthening the in vitro observations. These data provide further mechanistic insights linking JNK activation to the core apoptotic machinery during FFA-mediated lipotoxicity.DISCUSSIONThe principal findings of this study relate to the mechanisms of saturated FFA-mediated lipoapoptosis. Our results in Huh-7 cells and primary hepatocytes indicate that (i) the toxic saturated FFA palmitate, but not the monounsaturated FFA oleate, induces expression of PUMA mRNA and protein, (ii) saturated FFA-induced JNK1 activates and phosphorylates c-Jun, which directly binds the PUMA promoter and induces PUMA transcription, and (iii) genetic deletion or short hairpin RNA targeted knockdown of PUMA expression attenuates lipoapoptosis by palmitate. Our data also demonstrate that JNK is activated, and PUMA is up-regulated in the liver of patients with NASH. These observations suggest that PUMA up-regulation contributes to JNK1-potentiated cytotoxicity during lipogenic hepatocyte injury.Pro-apoptotic BH3-only protein family members are critical regulators of lipoapoptosis as small interfering RNA targeted knockdown of Bim partially protects liver cells against lipoapoptosis (12Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Although these findings indicate an important role for Bim in FFA-induced apoptosis, the partial protection also suggests that other BH3-only proteins with complementary functions contribute to lipotoxicity. The current study indicates that the saturated FFA palmitate increases PUMA expression in liver cells. In contrast to palmitate, the unsaturated FFA oleate minimally increases cellular PUMA protein levels, below the threshold sufficient to induce cell death (13Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 53Malhi H. Barreyro F.J. Isomoto H. Bronk S.F. Gores G.J. Gut. 2007; 56: 1124-1131Crossref PubMed Scopus (169) Google Scholar). These findings are consistent with prior observations that saturated FFA are inherently more toxic than unsaturated FFA (12Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 55Wei Y. Wang D. Topczewski F. Pagliassotti M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E275-E281Crossref PubMed Scopus (549) Google Scholar).Saturated FFA treatment also induced an increase in NOXA mRNA without affecting the expression of the other BH3-only proteins Bad, Bid, Bik, Bmf, and Hrk. However, the increase in NOXA mRNA did not cause an increase in NOXA protein expression. Whether this discordance is because of post-transcriptional modifications of NOXA by microRNAs, ribosomal occupation, or RNA sequestration remains to be explored. Also, as described for Bim (56Ley R. Balmanno K. Hadfield K. Weston C. Cook S.J. J. Biol. Chem. 2003; 278: 18811-18816Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), possible post-translational modifications (e.g. by phosphorylation, ubiquitination, etc.) of NOXA and further degradation by the proteasome pathway could shorten the half-life of the protein and mask a potential increase in NOXA protein translation. Given the fact that PUMA is a potent BH3-only protein (33Neise D. Graupner V. Gillissen B.F. Daniel P.T. Schulze-Osthoff K. Jänicke R.U. Essmann F. Oncogene. 2008; 27: 1387-1396Crossref PubMed Scopus (27) Google Scholar, 40Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 57Akhtar R.S. Geng Y. Klocke B.J. Latham C.B. Villunger A. Michalak E.M. Strasser A. Carroll S.L. Roth K.A. J. Neurosci. 2006; 26: 7257-7264Crossref PubMed Scopus (54) Google Scholar) which cooperates with Bim in cell death processes (43Erlacher M. Labi V. Manzl C. Böck G. Tzankov A. Häcker G. Michalak E. Strasser A. Villunger A. J. Exp. Med. 2006; 203: 2939-2951Crossref PubMed Scopus (189) Google Scholar), we focused our study on the contribution and the regulation of PUMA expression during lipoapoptosis.PUMA has been implicated in mediating cell death by a wide variety of toxic stimuli, including genotoxic stresses, serum withdrawal, or endoplasmic reticulum stress-inducing agents (39Jeffers J.R. Parganas E. Lee Y. Yang C. Wang J. Brennan J. MacLean K.H. Han J. Chittenden T. Ihle J.N. McKinnon P.J. Cleveland J.L. Zambetti G.P. Cancer Cell. 2003; 4: 321-328Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 58Luo X. He Q. Huang Y. Sheikh M.S. Cell Death Differ. 2005; 12: 1310-1318Crossref PubMed Scopus (55) Google Scholar). PUMA has been extensively studied in colon cancer cells, thymocytes, and neurons (32Ming L. Wang P. Bank A. Yu J. Zhang L. J. Biol. Chem. 2006; 281: 16034-16042Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 40Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 44Steckley D. Karajgikar M. Dale L.B. Fuerth B. Swan P. Drummond-Main C. Poulter M.O. Ferguson S.S. Strasser A. Cregan S.P. J. Neurosci. 2007; 27: 12989-12999Crossref PubMed Scopus (120) Google Scholar, 45Villunger A. Michalak E.M. Coultas L. Müllauer F. Böck G. Ausserlechner M.J. Adams J.M. Strasser A. Science. 2003; 302: 1036-1038Crossref PubMed Scopus (1081) Google Scholar, 59Michalak E.M. Villunger A. Adams J.M. Strasser A. Cell Death Differ. 2008; 15: 1019-1029Crossref PubMed Scopus (157) Google Scholar), but a potential contribution to liver injury has not been described. Our observations implicate PUMA in hepatic lipotoxicity. For example, short hairpin RNA-targeted knockdown of PUMA in Huh-7 cells or genetic deficiency of Puma in mouse primary hepatocytes renders liver cells partially resistant to FFA cytotoxicity. Saturated FFA induce apoptosis through the mitochondrial death pathway by activating Bax (13Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar), and PUMA promotes apoptosis in a Bax-dependent manner (32Ming L. Wang P. Bank A. Yu J. Zhang L. J. Biol. Chem. 2006; 281: 16034-16042Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 34Gallenne T. Gautier F. Oliver L. Hervouet E. Noël B. Hickman J.A. Geneste O. Cartron P.F. Vallette F.M. Manon S. Juin P. J. Cell Biol. 2009; 185: 279-290Crossref PubMed Scopus (128) Google Scholar, 60Chipuk J.E. Fisher J.C. Dillon C.P. Kriwacki R.W. Kuwana T. Green D.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20327-20332Crossref PubMed Scopus (193) Google Scholar, 61Jabbour A.M. Heraud J.E. Daunt C.P. Kaufmann T. Sandow J. O'Reilly L.A. Callus B.A. Lopez A. Strasser A. Vaux D.L. Ekert P.G. Cell Death Differ. 2009; 16: 555-563Crossref PubMed Scopus (62) Google Scholar). In agreement with these observations, our results suggest that PUMA contributes to Bax activation by saturated FFA, resulting in mitochondrial dysfunction, caspase 3/7 activation, and subsequent cell death.Whether PUMA directly or indirectly activates Bax is controversial. Recent studies have demonstrated a direct interaction between PUMA and the first α helix of Bax, which promotes Bax translocation to the mitochondria and triggers apoptosis (34Gallenne T. Gautier F. Oliver L. Hervouet E. Noël B. Hickman J.A. Geneste O. Cartron P.F. Vallette F.M. Manon S. Juin P. J. Cell Biol. 2009; 185: 279-290Crossref PubMed Scopus (128) Google Scholar). Also, PUMA may indirectly promote Bax activation by binding to pro-survival Bcl-2 proteins such as Mcl-1 and/or Bcl-XL, which are both expressed in hepatocytes (60Chipuk J.E. Fisher J.C. Dillon C.P. Kriwacki R.W. Kuwana T. Green D.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20327-20332Crossref PubMed Scopus (193) Google Scholar, 61Jabbour A.M. Heraud J.E. Daunt C.P. Kaufmann T. Sandow J. O'Reilly L.A. Callus B.A. Lopez A. Strasser A. Vaux D.L. Ekert P.G. Cell Death Differ. 2009; 16: 555-563Crossref PubMed Scopus (62) Google Scholar). By binding these anti-apoptotic proteins, PUMA may displace or prevent their sequestration of other BH3-only proteins such as Bim. The liberated Bim would then be able to directly activate Bax (62Yamaguchi H. Wang H.G. J. Biol. Chem. 2002; 277: 41604-41612Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Alternatively, binding of PUMA to an anti-apoptotic protein such as Bcl-XL can result in the dissociation of Bax from Bcl-XL, thereby indirectly promoting Bax mitochondrial activation (32Ming L. Wang P. Bank A. Yu J. Zhang L. J. Biol. Chem. 2006; 281: 16034-16042Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 61Jabbour A.M. Heraud J.E. Daunt C.P. Kaufmann T. Sandow J. O'Reilly L.A. Callus B.A. Lopez A. Strasser A. Vaux D.L. Ekert P.G. Cell Death Differ. 2009; 16: 555-563Crossref PubMed Scopus (62) Google Scholar). Detailed analysis of the mechanisms by which PUMA activates Bax in this model will require further studies.Although PUMA was originally identified as a p53 transcriptional target (40Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 41Nakano K. Vousden K.H. Mol. 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Whereas JNK2 appears to promote apoptosis in a tumor necrosis factor-induced liver injury model (66Kodama Y. Taura K. Miura K. Schnabl B. Osawa Y. Brenner D.A. Gastroenterology. 2009; 136: 1423-1434Abstract Full Text Full Text PDF PubMed Scopus (72) Google Schola
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