TGF-β enhances alcohol dependent hepatocyte damage via downregulation of alcohol dehydrogenase I

The adverse effect of alcohol in liver is mediated via interactions involving oxidative/non-oxidative metabolism, fat deposition and fibrogenic cytokines. This study assessed cross-talk between ethanol and TGF-β signaling in hepatocytes and its contribution to liver damage. To obtain a comprehensive view on TGF-β effects in hepatocytes, an Affymetrix microarray analysis was performed. Results were confirmed by qRT-PCR, IHC and Western blot. Cell toxicity was determined by lactate dehydrogenase assay. Neutral lipid deposition in hepatocytes was evaluated by OilRed-O staining. Small interfering RNA technique was used for gene silencing in vitro. TGF-β signaling is active and induced in alcohol dependent liver disease as showed by IHC staining for phosphorylated Smads. Ethanol and TGF-β, each display a dose-dependent cytotoxic effect on cultured mouse hepatocytes. The effect was further increased by combined treatment with ethanol and TGF-β. Microarray data implicate that TGF-β regulates expression of genes involved in fibrogenesis, lipid-, and oxidative stress metabolism. Interestingly, TGF-β decreased expression and activity of Alcohol Dehydogenase1 (ADH1). This down-regulation of ADH1 was also observed in vivo using samples from a transgenic mouse overexpressing active TGF-β. To delineate the TGF-β pathways regulating ADH1 expression, we identified the ALK5/Smad pathway which was inhibited by overexpression of antagonist Smad7. The profile of neutral lipid deposition from ethanol-fed ADH1-knockdown hepatocytes correlates with the development of a fatty liver condition. In correlation with these in vitro findings, ADH1 expression was decreased in an intragastric ethanol infusion mouse model. In summary our results support the hypothesis that TGF-β acts as pro-steatotic cytokine in ALD by downregulating the ethanol metabolizing enzyme ADH1, thereby decreasing hepatic clearance of ethanol which most likely contributes to increased alcohol-dependent liver damage.