Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking.

Alcoholic liver disease is a major biomedical health concern in the United States. Although the progression of this disease has been well described clinically, little is known about the molecular basis for liver injury. Research aimed at identifying the molecules and pathways that promote alcohol-induced hepatotoxicity has been hampered by the lack of good in vitro models. Our recent studies have been performed in WIF-B cells, an emerging model system for the study of alcohol-induced liver injury. These hepatic cells are highly differentiated and form discrete apical and basolateral membrane domains in culture. Domain-specific membrane proteins are localized in WIF-B cells as they are in hepatocytes in situ, and many liver-specific functions are maintained.1 Importantly, WIF-B cells metabolize ethanol using alcohol dehydrogenase (ADH) and cytochrome P4502E1.2 Like hepatocytes, ethanol-treated WIF-B cells display a reduced redox state and increased triglycerides.2 More recently, we discovered that microtubule repolymerization was impaired in ethanol-treated WIF-B cells,3 consistent with reports performed in vitro and in isolated hepatocytes.4–6 We also found that steady-state microtubules in alcohol-treated WIF-B cells were more stable and were acetylated 2–3-fold more than were those in control cells.3 This posttranslational modification is characteristic of stable microtubule populations (see Westermann and Weber7 and the Discussion section). We confirmed these results in livers from ethanol-fed rats, indicating the findings have physiologic importance.3 We further determined that increased microtubule acetylation and stability in WIF-B cells was dependent on ethanol metabolism and was likely mediated by acetaldehyde. Thus, alcohol consumption and metabolism alter hepatic microtubule dynamics. Because microtubules are central to multiple cellular processes, changes in their dynamics will likely alter hepatic function. This study was aimed at understanding the relationship between protein trafficking and alterations in microtubule dynamics. Not only is protein trafficking microtubule dependent, it is also selectively impaired by ethanol.8–10 Two transport pathways appear to be affected: transport of newly synthesized secretory or membrane proteins from the Golgi to the basolateral membrane and receptor-mediated endocytosis from the sinusoidal surface. Can the defects in secretion and endocytosis be explained by increased microtubule acetylation and stability? To answer this question, we examined the trafficking of selected proteins in WIF-B cells treated with ethanol or trichostatin A (TSA), an inhibitor of histone deacetylase 6 (HDAC6). HDAC6 is the major tubulin deacetylase in liver and WIF-B cells (Grozinger et al.12 and manuscript in preparation), and when inhibited with TSA, increased microtubule acetylation and stability is observed.13–15 We determined that 50 nM TSA for 30 minutes induced microtubule acetylation and stability to the same extent in WIF-B cells as did ethanol, allowing us to test our hypothesis. This result also suggested that acetylation induced microtubule stability, a topic that is currently controversial (for example, see Tran et al.15 and Palazzo et al.16 and the Discussion section). As shown in situ,8–10 ethanol impaired albumin secretion and internalization of asialoglycoprotein receptor (ASGP-R), further confirming that WIF-B cells are a good model for studying alcoholic liver injury. Similarly, TSA treatment impaired albumin secretion and ASGP-R internalization, but to a lesser extent. Both agents impaired internalization of the single-spanning apical resident aminopeptidase N (APN), but distribution of the glycophosphatidylinositol (GPI)–anchored protein 5′-nucleotidase (5′-NT) was not altered, suggesting that microtubule acetylation and stability selectively regulate internalization. We concluded that increased microtubule acetylation and stability in part explain the alcohol-induced defects in membrane trafficking.