Role of Sumoylation in Alcoholic Liver Disease

描述（由申请人提供）：酒精滥用是肝病死亡率的主要因素，并增加了广泛的不良健康影响的风险。肝脏作为酒精代谢的主要部位，是酒精损伤的主要靶点。酒精性肝病（ALD）的范围包括单纯性脂肪变性、酒精性肝炎、纤维化、肝硬化和肝细胞癌。类小泛素化是一种翻译后修饰，可调节多种细胞过程，如信号转导、应激反应、细胞运输、蛋白质-蛋白质相互作用、蛋白质-DNA相互作用和转录活性。 SUMO由四种不同的蛋白质组成（SUMO-1，-2，3-和-4）。SUMO化通常在氧化应激下增加。我们最近报道了泛素结合酶9（ubiquitin conjugating enzyme 9，Ubc 9），sumoylation的唯一E2酶，在胃内乙醇灌注（EI）处理的小鼠中被诱导，但其功能意义尚不清楚。因此，我们发现SUMO-1，-3和Ubc 9 mRNA在EI小鼠肝脏中的表达增加。此外，EI小鼠显示SUMO-1引起的蛋白质类小泛素化的总体增加，但SUMO-2/3引起的类小泛素化仅发生微小变化。原代小鼠肝细胞的乙醇处理导致活性氧（ROS）和甘油三酯产生增加。此外，我们发现Ubc 9和SUMO基因，Cyp 2 e1的表达增加，SUMO-1蛋白SUMO化的总体增加，如EI肝脏。Ubc 9的沉默阻止了乙醇诱导的脂肪积累，ROS的产生和原代小鼠肝细胞中Cyp 2 e1表达的增加。在LX-2细胞（活化的人肝星状细胞或HSC）中，乙醇处理也增加了Ubc 9表达、ROS产生和HSC活化标志物。阻断Ubc 9诱导可阻止所有这些并诱导HSC凋亡。最后，我们发现脂多糖（LPS）和Ubc 9 RNAi单独处理增加RAW细胞（巨噬细胞系）中促炎细胞因子的表达;但是当LPS和Ubc 9 RNAi组合时，这些细胞因子的表达进一步增加。有趣的是，LPS处理降低了Ubc 9蛋白水平（mRNA水平不变）。这一提议正在验证一个新的假设，即在类小泛素化中存在调节异常，其以细胞类型特异性的方式促成ALD的发病机制。提出了三个具体的目的来检查：1）sumoylation在乙醇诱导的肝细胞变化中的作用，2）sumoylation在乙醇诱导的HSC活化中的作用，以及3）sumoylation在ALD中枯否细胞活化中的作用。如果成功完成，这些研究应该提供高度新颖的信息sumoylation在ALD的发展中的作用，并可能提供新的治疗策略，这是高度优先的公共卫生。

DESCRIPTION (provided by applicant): Alcohol abuse is a leading factor in mortality from liver disease and increases the risk for a wide range of adverse health effects. The liver, as the primary site of alcohol metabolism, is a major target of injury. The spectrum of Alcoholic Liver Diseases (ALD) includes simple steatosis, alcoholic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Sumoylation is a post-translational modification that modulates multiple cellular processes such as signal transduction, stress responses, cellular trafficking, protein-protein interactions, protein-DNA interactions and transcriptional activity. SUMO is comprised of four distinct proteins in humans (SUMO-1, -2, 3- and -4). Sumoylation is often increased under oxidative stress. We recently reported that ubiquitin conjugating enzyme 9 (Ubc9), the sole E2 enzyme of sumoylation, is induced in intragastric ethanol- infusion (EI) treated mice but the functional significance of this is unknown. Consistently, we found SUMO-1, -3 and Ubc9 mRNA levels are increased the livers of EI mice. Also, EI mice show an overall increase in protein sumoylation by SUMO-1 but only minor changes in sumoylation by SUMO-2/3. Ethanol treatment of primary mouse hepatocytes leads to increased reactive oxygen species (ROS) and triglyceride production. In addition, we found increased expression of Ubc9 and SUMO genes, Cyp2e1 and an overall increase in SUMO-1 protein sumoylation like in EI livers. Silencing of Ubc9 prevented ethanol-induced fat accumulation, ROS production and increased Cyp2e1 expression in primary mouse hepatocytes. In LX-2 cells (activated human hepatic stellate cells or HSCs), ethanol treatment also increased Ubc9 expression, ROS production and HSC activation markers. Blocking Ubc9 induction prevented all of these and induced apoptosis in HSCs. Finally, we found that lipopolysaccharide (LPS) and Ubc9 RNAi treatment alone increased expression of proinflammatory cytokines in RAW cells (macrophage cell line); but when LPS and Ubc9 RNAi were combined, the expression of these cytokines increased further. Interestingly, LPS treatment decreased Ubc9 protein level (mRNA level was unchanged). This proposal is testing the novel hypothesis that there is dysregulation in sumoylation that contributes to the pathogenesis of ALD in a cell-type specific manner. Three specific aims are proposed to examine: 1) the role of sumoylation in ethanol-induced changes in hepatocytes, 2) the role of sumoylation in ethanol-induced HSC activation, and 3) the role of sumoylation in Kupffer cell activation in ALD. If successfully completed, these studies should provide highly novel information on the role of sumoylation in the development of ALD and may provide novel therapeutic strategies, which is of high public health priority.