Role of the life span determinant P66shcA in ethanol-induced liver damage
Role of the life span determinant P66shcA in ethanol-induced liver damage
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DOI：
10.1038/labinvest.2008.44
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发表时间：
2008-07-01
影响因子：
5
通讯作者：
Pani, Giovambattista
中科院分区：
文献类型：
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作者：
Koch, Osvaldo R.;Fusco, Salvatore;Pani, Giovambattista
缺乏适配器分子shcA （p66(shcA)）的66 kDa异构体的小鼠显示出对氧化应激的抵抗力增强和延迟衰老。在培养的细胞系中，p66促进线粒体中活性氧（ROS）的形成，并在各种促氧化有害刺激下促进凋亡细胞死亡。由于线粒体ROS和氧化细胞损伤明显参与酒精诱导的病理，我们假设p66也可能在乙醇中起作用。在体内，p66+/+小鼠在饮用水中暴露乙醇6周后观察到的变化，包括血清丙氨酸转氨酶（ALT）升高、肝脏肿胀和明显的肝脏脂肪变性，在p66-/-突变小鼠中显著减弱。肝脏组织的生化分析显示乙醇诱导了p66蛋白，而p66缺乏的肝脏对酒精的反应是线粒体抗氧化酶MnSOD的显著上调，而在对照组小鼠中几乎没有。在体外原代肝细胞和HepG2-E47细胞（一种乙醇反应性肝癌细胞系）中，p66的表达水平与酒精诱导的氧化应激保护之间也存在负相关的证据。事实上，与野生型分离肝细胞相比，暴露于乙醇的MnSOD上调在p66KO中更为明显，而在过表达p66shc的HepG2细胞中则减弱。p66过表达也阻止了由SOD2启动子控制的荧光素酶报告基因的激活，这表明p66抑制MnSOD在转录水平上起作用。最后，p66在HepG2细胞中产生ROS，并通过乙醇增强氧化应激和线粒体去极化。综上所述，上述观察清楚地表明p66在酒精诱导的细胞损伤中的作用，可能是通过细胞自主机制，涉及抗氧化防御表达减少和线粒体功能障碍。
Mice lacking the 66 kDa isoform of the adapter molecule shcA (p66(shcA)) display increased resistance to oxidative stress and delayed aging. In cultured cell lines, p66 promotes formation of Reactive Oxygen Species (ROS) in mitochondria, and apoptotic cell death in response to a variety of pro-oxidant noxious stimuli. As mitochondrial ROS and oxidative cell damage are clearly involved in alcohol-induced pathology, we hypothesized that p66 may also have a role in ethanol. In vivo, changes observed in p66+/+ mice after 6-week exposure to ethanol in the drinking water, including elevated serum alanine aminotransferase (ALT), liver swelling and evident liver steatosis, were significantly attenuated in p66-/- mutant mice. Biochemical analysis of liver tissues revealed induction of the p66 protein by ethanol, whereas p66-deficient livers responded to alcohol with a significant upregulation of the mitochondrial antioxidant enzyme MnSOD, nearly absent in control mice. Evidence of an inverse correlation between expression level of p66 and protection from alcohol-induced oxidative stress was also confirmed in vitro in primary hepatocytes and in HepG2-E47 cells, an ethanol-responsive hepatoma cell line. In fact, MnSOD upregulation by exposure to ethanol in vitro was much more pronounced in p66KO versus wild-type isolated liver cells, and blunted in HepG2 cells overexpressing p66shc. p66 overexpression also prevented the activation of a luciferase reporter gene controlled by the SOD2 promoter, indicating that p66 repression of MnSOD operates at a transcriptional level. Finally, p66 generated ROS in HepG2 cells and potentiated oxidative stress and mitochondrial depolarization by ethanol. Taken together, the above observations clearly indicate a role for p66 in alcohol-induced cell damage, likely via a cell-autonomous mechanism involving reduced expression of antioxidant defenses and mitochondrial dysfunction.