Chronic alcohol abuse and alcoholism are associated with high morbidity and mortality and known to cause major health problems such as alcoholic liver disease. Altered protein trafficking and glycosylation, and increased apoptosis have been reported in ethanol-exposed liver cells. But the mechanism remains unresolved. Recently, we found that non-muscle myosin IIA (NMIIA), a motor protein, interacts with the cytoplasmic tail of Golgi glycosyltransferases (GT) to induce Golgi fragmentation in cells under stress. The Golgi fragmentation was detected in hepatocytes exposed to alcohol in vitro and in vivo. Alcohol metabolites are responsible for this effect. Alcohol treatment also increases Rab6A GTPase, NMIIA, caspase-3 activity, NMIIA-GT complexes but decreases Golgi matrix protein, Giantin, and GT. In control cells, knockdown of Giantin retains GT in the endoplasmic reticulum. Knockdown of NMIIA or Rab6A prevents alcohol treatment- induced Golgi fragmentation. The results suggest that NMIIA and Rab6A are intimately involved in Golgi fragmentation induced by alcohol treatment. Further, the reduction of GT induced by alcohol treatment could be explained by (a) its elevated Golgi-to-endoplasmic reticulum retrograde transport forced by increased NMIIA-GT complexes coupled with (b) its impaired Golgi targeting resulted from elevated degradation of Giantin caused by activated caspase-3 activity. We propose to test the hypothesis that alcohol treatment- induced Golgi fragmentation is responsible for reduced glycosylation and function of asialoglycoprotein receptors as well as induction of apoptosis. The four specific aims of the proposed study are to: 1. Examine how during alcohol-specific Golgi fragmentation Rab6A regulates the interaction of NMIIA with GT followed by increased NMIIA-GT complexes;2. Examine how elevated caspases-3 activity induced by alcohol treatment impairs ER-to-Golgi transport of GT;3. Determine how the alcohol treatment-induced Golgi fragmentation affects glycan structure and function of asialoglycoprotein receptors including apoptosis;and 4. Validate the results of specific aims 1-3 obtained in VA-13 cells in the hepatocytes of alcohol-treated mice with and without functional asialoglycoprotein receptors. Accomplishment of the goal of the proposed study would expand our understanding of the regulation of cell death caused by ethanol abuse and help identify potential targets for developing therapy to treat alcoholic liver disease.
Chronic alcohol abuse can increase apoptosis of hepatocytes, which leads to liver injury and alcohol liver disease. Current application will elucidate how alcohol treatment-induced Golgi fragmentation results in elevated apoptosis of hepatocytes. The results could help identify the molecules/events that are responsible for the development of alcoholic liver apoptosis, which could lead to development of mechanism-based therapy to treat alcohol liver disease.
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