Z Gastroenterol 2009; 47 - P327
DOI: 10.1055/s-0029-1241575

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

LI Ciuclan 1, S Ehnert 1, I Ilkavets 1, H Weng 1, H Gaitantzi 1, H Tsukamoto 2, E Ueberham 1, MV Singer 1, K Breitkopf 1, S Dooley 1
  • 1Universitätsmedizin Mannheim der Universität Heidelberg, II. Medizinische Klinik, Molekulare Gastroenterologie – Alkoholfolgeerkrankungen, Mannheim, Germany
  • 2School of Medicin of USC, MMR 412, Los Angeles, United States

Background/aims: The adverse effects of alcohol in liver is mediated via complex 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.

Methods: Affymetrix microarray analysis was performed to examine the TGF-β effects in hepatocytes. Results were confirmed by qRT-PCR Western blot and Immunohistochemistry (IHC). Cell damage was determined by lactate dehydrogenase (LDH) assay and oxidative balance was analysed by measuring the cellular glutathione (GSH), reactive oxygen species (ROS) and lipid peroxidation levels. Neutral lipid deposition in hepatocytes was evaluated by OilRed-O staining. siRNA technique was used for gene silencing in vitro.

Results: As shown by IHC for phosphorylated Smads in liver biopsy samples from mice TGF-β signaling is active and induced in alcohol dependent liver disease. Ethanol and TGF-β, each display a dose-dependent cytotoxic effect on cultured mouse hepatocytes as shown by release of LDH and production of ROS. 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 as well as 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 over-expressing active TGF-β. The profile of neutral lipid deposition in hepatocytes from ethanol-fed ADH1-knockdown mice correlates with the development of a fatty liver. To delineate the TGF-β pathways regulating ADH1 expression, we identified the ALK5/Smad pathway which was inhibited by over-expression of the TGF-β antagonist Smad7. In correlation with these in vitro findings, ADH1 expression was decreased in an intragastric ethanol infusion mouse model as shown by IHC.

Conclusion: In summary our in vitro and in vivo results support the hypothesis that TGF-β acts as pro-steatotic cytokine in ALD by down-regulating the ethanol metabolizing enzyme ADH1, thereby decreasing hepatic clearance of ethanol which most likely contributes to increased alcohol-dependent liver damage.