Role of Sumoylation in Alcoholic Liver Disease

描述(由申请人提供)：酗酒是肝病死亡率的主要因素，并增加了一系列不利健康影响的风险。肝脏作为酒精代谢的主要部位，是损伤的主要靶点。酒精性肝病(ALD)包括单纯性脂肪变性、酒精性肝炎、纤维化、肝硬变和肝细胞癌。苏莫化是一种翻译后修饰，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。相扑在人类中由四种不同的蛋白质组成(相扑-1、相扑2、相扑3和相扑4)。在氧化应激下，相思甲基化通常会增加。我们最近报道在乙醇灌胃(EI)处理的小鼠中，唯一的E2酶--泛素结合酶9(Ubc9)被诱导，但其功能意义尚不清楚。我们一致地发现，相扑-1、-3和Ubc9的mRNA水平在EI小鼠的肝脏中增加。此外，EI小鼠表现出SUMO-1蛋白总和甲基化的增加，但SUMO-2/3只有微小的变化。乙醇处理原代小鼠肝细胞导致活性氧自由基(ROS)和甘油三酯产生增加。此外，我们发现在EI肝脏中Ubc9和SUMO基因、CYP2E1的表达增加，并且SUMO-1蛋白的总和甲基化增加。沉默Ubc9可阻止乙醇诱导的原代小鼠肝细胞脂肪堆积、ROS产生和CYP2E1表达增加。在LX-2细胞(活化的人肝星状细胞或HSCs)中，乙醇处理也增加了Ubc9的表达、ROS的产生和HSC的激活标志物。阻断Ubc9的诱导可阻止上述过程，并诱导HSCs的凋亡。最后，我们发现脂多糖(LPS)和Ubc9 RNAi单独作用能增加巨噬细胞系RAW细胞中促炎症细胞因子的表达，但当LPS和Ubc9 RNAi联合作用时，这些细胞因子的表达进一步增加。有趣的是，内毒素处理降低了Ubc9蛋白水平(信使核糖核酸水平不变)。这一建议是在检验一种新的假设，即在细胞类型特异性的方式中，存在相思作用失调，从而导致ALD的发病。我们提出了三个特定的目标：1)苏莫化在乙醇诱导的肝细胞改变中的作用；2)苏莫化在乙醇诱导的HSC激活中的作用；3)苏莫化在酒精性肝病中库普弗细胞激活中的作用。如果成功完成，这些研究将提供有关相思甲基化在ALD发展中的作用的高度新颖的信息，并可能提供新的治疗策略，这是高度公共卫生优先考虑的。

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.