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.