Cazanave S C, Wang X, Zhou H, Rahmani M, Grant S, Durrant D E, Klaassen C D, Yamamoto M, Sanyal A J
Department of Internal Medicine, Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
Cell Death Differ. 2014 Aug;21(8):1303-12. doi: 10.1038/cdd.2014.49. Epub 2014 Apr 25.
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.
非酒精性脂肪性肝炎的特征是肝脂肪变性、循环游离脂肪酸（FFA）水平升高和肝细胞脂肪凋亡。这种脂肪凋亡需要增加JNK磷酸化以及促凋亡的仅含BH3结构域蛋白Bim和PUMA的激活。 Kelch样ECH相关蛋白（Keap）-1是一种BTB/Kelch蛋白，可调节Bcl-2蛋白的表达并控制凋亡细胞死亡。然而，Keap1在肝细胞脂肪毒性中的作用尚不清楚。在这里，我们证明，在对毒性饱和脂肪酸棕榈酸的反应中，Keap1蛋白在肝细胞中通过自噬以p62依赖的方式迅速降解，但在用无毒脂肪酸油酸孵育后则不会。使用shRNA技术在肝癌细胞系中稳定敲低Keap1表达可诱导自发细胞毒性，这与JNK1依赖的Bim和PUMA蛋白水平上调有关。此外，敲低Keap1可使肝细胞对棕榈酸诱导的脂肪凋亡更加敏感。同样，从肝脏特异性Keap1（-/-）小鼠分离的原代肝细胞显示出更高的Bim和PUMA蛋白水平，并且比野生型小鼠肝细胞对棕榈酸诱导的凋亡表现出更高的敏感性。最后，稳定敲低Bim或PUMA表达可防止因Keap1缺失诱导的细胞毒性。这些结果表明，棕榈酸通过p62依赖的自噬降解Keap1是导致肝细胞脂肪凋亡的一种机制。
