Chronic Alcohol Consumption Impairs Clathrin-Mediated Endocytosis and Microtubule-Dependent Nuclear Translocation in Hepatocytes
The molecular basis for alcohol-induced hepatotoxicity is not well-understood. We sought to further explore a known protein trafficking defect caused by chronic alcohol exposure: receptor internalization from the hepatocyte plasma membrane, and a hypothesized defect in microtubule-dependent nuclear translocation of transcription factors. We hypothesized that both of these defects are caused by ethanol-induced protein hyperacetylation. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor (ASGP-R) was impaired in ethanol-treated WIF-B cells, whereas the internalization of a glycophosphatidylinositol (GPI)-anchored protein internalized via a caveolae/raft-mediated pathway was not changed. ASGP-R internalization was also impaired by trichostatin-A (TSA), a drug that induces global protein hyperacetylation, providing evidence that hyperacetylation is indeed associated with this defect. We examined a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. Markers known to be internalized via clathrin-mediated endocytosis were impaired, whereas the internalization of markers for caveolae/raft-mediated-, fluid phase-, or non-vesicle-mediated mechanisms was not altered after ethanol exposure. We conclude that clathrin-mediated endocytosis is selectively impaired by ethanol exposure. Previously we found that alcohol exposure led to increased microtubule acetylation and stability in WIF-B cells and livers from ethanol-fed rats. Because dynamic microtubules are known to regulate nuclear translocation of some transcription factors, we examined whether alcohol-induced microtubule acetylation and stability impair nuclear translocation. Representing factors that undergo directed nuclear delivery, we examined the growth hormone-induced translocation of Signal Transducer and Activator of Transcription 5B (STAT5B) and the interleukin 6 (IL-6) -induced translocation of STAT3. Representing factors that are sequestered in the cytoplasm by microtubule attachment until ligand activation, we examined the Transforming Growth Factor-Beta (TGF-Beta) -induced translocation of Smad2/3. Ethanol exposure impaired the translocation of STAT3 and STAT5B, but not Smad2/3. STAT5B translocation was decreased to similar extents by taxol or trichostatin A, agents that promote microtubule acetylation in the absence of alcohol. Thus, the alcohol-induced impairment of STAT nuclear translocation can be explained by increased microtubule acetylation. Only ethanol-treatment impaired STAT5B activation, indicating microtubule acetylation is not important for this process. Together, these results suggest that deacetylase agonists may be effective therapeutics for treating alcoholic liver disease.
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