Transcriptional Regulation of Bim By FOXO3A Mediates Hepatocytes

FERNANDO J BARREYRO; SHOGO KOBAYASHI; STEVEN F BRONK; NATHAN W. WERNEBURG; HARMEET MALHI; GREGORY J GORES

Washington DC, USA

Congreso; DDW-2007 Annual Meeting of the American Gastroenterological Association (AGA). 2007; 2007

American Gastroenterological Association

  Hepatocyte lipoapoptosis, a critical feature of non-alcoholic steatohepatitis, can be replicated In Vitro by incubating hepatocytes with saturated free fatty acids (FFA). These toxic FFA induce expression of Bim, a proapoptotic, BH3-only protein of the Bcl-2 family. Although Bim expression is essential for lipoapoptosis, the mechanisms by which FFA induce Bim remains unclear. Because the forkhead box, class O 3a (FoxO3a), transcription factor has been implicated in Bim expression, our aim was to test the hypothesis that FFA induce Bim by a FoxO3a-dependent mechanism. METHODS: Lipoapoptosis was induced by treating the human cell lines, Huh7 and HepG2 cells with the saturated FFA stearic acid (SA) and palmitic acid (PA), and the monounsaturated FFA oleic acid (OA). Bim expression was assessed by immunoblot analysis and quantitative real time PCR (QRT-PCR). To determine if FoxO3a binds to the Bim promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Knockdown of FoxO3a was achieved by siRNA technology, and apoptosis assessed by characteristic nuclear staining with DAPI and caspase 3/7 activity. RESULTS: The saturated FFA, PA and SA (400 ìM for 6 hrs) increased Bim mRNA expression 16- fold, while OA only increased Bim expression 3-fold. Comparable fold changes in cellular Bim protein levels were also observed following treatment with these FFA. FoxO3a is normally inactive in cells as a phosphoprotein complex to protein 14-3-3 in the cytosol; upon activation it becomes dephosphorylated and translocates to the nucleus. Consistent with this paradigm, PA and SA, but not OA, induced significant FoxO3a dephosphorylation and nuclear translocation as assessed by phosphoimmunoblot analysis and cellular immunofluorescence, respectively. Direct binding of FoxO3a to the Bim promoter following incubation with PA or SA was confirmed by the ChIP assay. Moreover, the saturated FFA also directly activated a FoxO luciferase-based reporter assay. Finally, we assessed the role of FoxO3a-mediated Bim expression in FFA-induced lipoapoptosis. A siRNA targeted knockdown of FoxO3a was confirmed by immunoblot analysis. In the presence of FoxO3a siRNA, SA and PA induced Bim expression was abrogated. Furthermore FFA treated FoxO3a siRNA transfected cells demonstrated greater than a 50% reduction in lipoapoptosis. In CONCLUSION, saturated FFA induce hepatocyte lipoapoptosis, in part, by transcriptionally regulating the cell death protein Bim. Modulation of this pathway may be a potential therapeutic strategy for non-alcoholic fatty liver injury. the human cell lines, Huh7 and HepG2 cells with the saturated FFA stearic acid (SA) and palmitic acid (PA), and the monounsaturated FFA oleic acid (OA). Bim expression was assessed by immunoblot analysis and quantitative real time PCR (QRT-PCR). To determine if FoxO3a binds to the Bim promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Knockdown of FoxO3a was achieved by siRNA technology, and apoptosis assessed by characteristic nuclear staining with DAPI and caspase 3/7 activity. RESULTS: The saturated FFA, PA and SA (400 ìM for 6 hrs) increased Bim mRNA expression 16- fold, while OA only increased Bim expression 3-fold. Comparable fold changes in cellular Bim protein levels were also observed following treatment with these FFA. FoxO3a is normally inactive in cells as a phosphoprotein complex to protein 14-3-3 in the cytosol; upon activation it becomes dephosphorylated and translocates to the nucleus. Consistent with this paradigm, PA and SA, but not OA, induced significant FoxO3a dephosphorylation and nuclear translocation as assessed by phosphoimmunoblot analysis and cellular immunofluorescence, respectively. Direct binding of FoxO3a to the Bim promoter following incubation with PA or SA was confirmed by the ChIP assay. Moreover, the saturated FFA also directly activated a FoxO luciferase-based reporter assay. Finally, we assessed the role of FoxO3a-mediated Bim expression in FFA-induced lipoapoptosis. A siRNA targeted knockdown of FoxO3a was confirmed by immunoblot analysis. In the presence of FoxO3a siRNA, SA and PA induced Bim expression was abrogated. Furthermore FFA treated FoxO3a siRNA transfected cells demonstrated greater than a 50% reduction in lipoapoptosis. In CONCLUSION, saturated FFA induce hepatocyte lipoapoptosis, in part, by transcriptionally regulating the cell death protein Bim. Modulation of this pathway may be a potential therapeutic strategy for non-alcoholic fatty liver injury. induce expression of Bim, a proapoptotic, BH3-only protein of the Bcl-2 family. Although Bim expression is essential for lipoapoptosis, the mechanisms by which FFA induce Bim remains unclear. Because the forkhead box, class O 3a (FoxO3a), transcription factor has been implicated in Bim expression, our aim was to test the hypothesis that FFA induce Bim by a FoxO3a-dependent mechanism. METHODS: Lipoapoptosis was induced by treating the human cell lines, Huh7 and HepG2 cells with the saturated FFA stearic acid (SA) and palmitic acid (PA), and the monounsaturated FFA oleic acid (OA). Bim expression was assessed by immunoblot analysis and quantitative real time PCR (QRT-PCR). To determine if FoxO3a binds to the Bim promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Knockdown of FoxO3a was achieved by siRNA technology, and apoptosis assessed by characteristic nuclear staining with DAPI and caspase 3/7 activity. RESULTS: The saturated FFA, PA and SA (400 ìM for 6 hrs) increased Bim mRNA expression 16- fold, while OA only increased Bim expression 3-fold. Comparable fold changes in cellular Bim protein levels were also observed following treatment with these FFA. FoxO3a is normally inactive in cells as a phosphoprotein complex to protein 14-3-3 in the cytosol; upon activation it becomes dephosphorylated and translocates to the nucleus. Consistent with this paradigm, PA and SA, but not OA, induced significant FoxO3a dephosphorylation and nuclear translocation as assessed by phosphoimmunoblot analysis and cellular immunofluorescence, respectively. Direct binding of FoxO3a to the Bim promoter following incubation with PA or SA was confirmed by the ChIP assay. Moreover, the saturated FFA also directly activated a FoxO luciferase-based reporter assay. Finally, we assessed the role of FoxO3a-mediated Bim expression in FFA-induced lipoapoptosis. A siRNA targeted knockdown of FoxO3a was confirmed by immunoblot analysis. In the presence of FoxO3a siRNA, SA and PA induced Bim expression was abrogated. Furthermore FFA treated FoxO3a siRNA transfected cells demonstrated greater than a 50% reduction in lipoapoptosis. In CONCLUSION, saturated FFA induce hepatocyte lipoapoptosis, in part, by transcriptionally regulating the cell death protein Bim. Modulation of this pathway may be a potential therapeutic strategy for non-alcoholic fatty liver injury. the human cell lines, Huh7 and HepG2 cells with the saturated FFA stearic acid (SA) and palmitic acid (PA), and the monounsaturated FFA oleic acid (OA). Bim expression was assessed by immunoblot analysis and quantitative real time PCR (QRT-PCR). To determine if FoxO3a binds to the Bim promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Knockdown of FoxO3a was achieved by siRNA technology, and apoptosis assessed by characteristic nuclear staining with DAPI and caspase 3/7 activity. RESULTS: The saturated FFA, PA and SA (400 ìM for 6 hrs) increased Bim mRNA expression 16- fold, while OA only increased Bim expression 3-fold. Comparable fold changes in cellular Bim protein levels were also observed following treatment with these FFA. FoxO3a is normally inactive in cells as a phosphoprotein complex to protein 14-3-3 in the cytosol; upon activation it becomes dephosphorylated and translocates to the nucleus. Consistent with this paradigm, PA and SA, but not OA, induced significant FoxO3a dephosphorylation and nuclear translocation as assessed by phosphoimmunoblot analysis and cellular immunofluorescence, respectively. Direct binding of FoxO3a to the Bim promoter following incubation with PA or SA was confirmed by the ChIP assay. Moreover, the saturated FFA also directly activated a FoxO luciferase-based reporter assay. Finally, we assessed the role of FoxO3a-mediated Bim expression in FFA-induced lipoapoptosis. A siRNA targeted knockdown of FoxO3a was confirmed by immunoblot analysis. In the presence of FoxO3a siRNA, SA and PA induced Bim expression was abrogated. Furthermore FFA treated FoxO3a siRNA transfected cells demonstrated greater than a 50% reduction in lipoapoptosis. In CONCLUSION, saturated FFA induce hepatocyte lipoapoptosis, in part, by transcriptionally regulating the cell death protein Bim. Modulation of this pathway may be a potential therapeutic strategy for non-alcoholic fatty liver injury.