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.
Sumoylation is a modification found in mammalian proteins due to which a SUMO group is tagged onto them. This regulates the function and fate of a large number of proteins involved in many important cellular functions including transcription, intracellular transport, DNA repair, replication, and cell signaling. This project aims to examine mechanism(s) of changes in sumoylation and functional outcome in Alcoholic Liver Diseases (ALD). Given that ALD is the major cause of liver disease in Western countries, if the proposed studies are successfully accomplished, these results should provide highly novel information on the role of sumoylation in the development of ALD.
