Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis.
Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1(-/-) mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis.
Department of Internal Medicine, Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Cazanave
Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Wang
Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Zhou
Department of Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Rahmani
Department of Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Grant
Department of Internal Medicine, Division of Cardiology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Durrant
Department of Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
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Klaassen
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
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Yamamoto
Department of Internal Medicine, Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
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Sanyal
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins; C488588:PUMA protein, mouse; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins