Alcohol-induced alterations in hepatic microtubule dynamics can be explained by impaired histone deacetylase 6 function

Abstract We have been using polarized, hepatic WIF-B cells to examine ethanol-induced liver injury. These cells polarize in culture and maintain numerous liver-specific activities including the ability to metabolize alcohol. Previously, we found that microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells and that increased microtubule acetylation required ethanol metabolism and was likely mediated by acetaldehyde. This study was aimed at identifying the mechanism responsible for increased microtubule acetylation. We examined the expression of two known microtubule deacetylases, histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2), in WIF-B cells. Immunoblotting, immunofluorescence microscopy, and assays using the SirT2 inhibitor nicotinamide revealed that WIF-B cells do not express SirT2. In contrast, HDAC6 was highly expressed in WIF-B cells. Addition of trichostatin A (TSA), an HDAC6 inhibitor, induced microtubule acetylation to the same extent as in ethanol-treated cells (approximately threefold). Although immunofluorescence labeling revealed that HDAC6 distribution did not change in ethanol-treated cells, immunoblotting showed HDAC6 protein levels slightly decreased. HDAC6 solubility was increased in nocodazole-treated cells, suggesting impaired microtubule binding. Direct microtubule binding assays confirmed this hypothesis. The decreased microtubule binding was partially prevented by 4-methyl pyrazole, indicating the effect was in part mediated by acetaldehyde. Interestingly, HDAC6 from ethanol-treated cells was able to bind and deacetylate exogenous tubulin to the same extent as control, suggesting that ethanol-induced tubulin modifications prevented HDAC6 binding to endogenous microtubules. Conclusion: We propose that lower HDAC6 levels combined with decreased microtubule binding lead to increased tubulin acetylation in ethanol-treated cells. (Hepatology 2008.)