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

The liver is the major site of ethanol metabolism and thus sustains the most injury from chronic alcohol consumption. Although the progression of alcoholic liver disease is well described, little is known about the molecular mechanisms that promote alcohol-induced hepatotoxicity. Our studies have been performed in WIF-B cells, an emerging model system for studying liver injury. These cells polarize in culture and exhibit many liver-specific activities.1 Importantly, WIF-B cells metabolize alcohol using endogenous alcohol dehydrogenase, acetaldehyde dehydrogenase, and cytochrome P450 2E1.2 Recently, we determined that microtubules were more stable and hyperacetylated in ethanol-treated WIF-B cells.3 This phenotype required alcohol metabolism and was likely mediated by acetaldehyde. We also determined that microtubules were acetylated to the same extent in livers from ethanol-fed rats, indicating the effect has physiological relevance.3 However, the mechanism responsible for the ethanol-induced tubulin hyperacetylation and increased stability is not known. Protein acetylation results from the coordinated activities of acetyltransferases and deacetylases.4,5 Although α–tubulin is known to be acetylated on lysine 40, the identity of the acetyltransferase is not known.6 However, there are two known microtubule deacetylases, histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2).7–10 These enzymes are part of a larger family of at least 18 members known collectively as histone deacetylases.11 The family is divided into three classes based on sequence identity and co-factor/co-enzyme dependence. Although most function in the nucleus to deacetylate histones and other transcriptional machinery,11,12 HDAC6 (class II) and SirT2 (class III) are cytosolic deacetylases that colocalize with microtubules in some cell types.7–10 Overexpression of either enzyme led to a loss of acetylated microtubules and decreased microtubule stability.7–10 Conversely, when the deacetylases were inactivated or their expression knocked down, microtubules were hyperacetylated and more stable.7–10 Because HDAC6 is enriched in liver13,14 (its expression in hepatic cell lines and isolated hepatocytes has not been examined) and because class III enzymes, including SirT2, require the oxidized form of reduced nicotinamide adenine dinucleotide for activity (the oxidized form of reduced nicotinamide adenine dinucleotide is a coenzyme for alcohol dehydrogenase and acetaldehyde dehydrogenase),9 both HDAC6 and SirT2 are good candidates for mediating alcohol-induced microtubule hyperacetylation. We found that WIF-B cells do not express SirT2, consistent with reports that SirT2 expression is enriched in brain.15,16 However, HDAC6 was abundantly expressed in WIF-B cells, and its inhibition with trichostatin A (TSA) led to increased microtubule acetylation comparable to that observed in ethanol-treated cells. Although alcohol or TSA did not alter HDAC6 subcellular distribution, it led to decreased HDAC6 protein levels. Furthermore, HDAC6 binding to microtubules was significantly impaired in ethanol-treated cells. HDAC6 from ethanol-treated cells bound to and deacetylated exogenous tubulin to the same extent as HDAC6 from control cells. Thus, impaired binding of HDAC6 to microtubules is likely attributable to ethanol-induced tubulin modifications that prevented associations. Together these results indicate that the alcohol-induced increases in microtubule acetylation and stability in WIF-B cells were attributable, in part, to decreased HDAC6 protein levels and decreased microtubule binding.