Interestingly, the TGF-b1-induced EMT morphology was robustly enhanced by Smad7 deletion. We also analyzed the cell motility using standard scratch-wound assays as previously described. At 48 h after wounding, the untreated cells from both wild type and Smad7-deleted mice were unable to migrate into the wound area. TGF-b1 treatment was able to induce migration of the cells and such effect was significantly accelerated when Smad7 was deleted. TGF-b-induced EMT was further analyzed by immunoblotting to detect expression of E-cadherin and vimentin, two well-recognized markers for EMT. We found that TGF-b1-induced reduction of E-cadherin and increase of vimentin was profoundly enhanced by Smad7 deletion. These data, therefore, reveal that Smad7 deficiency is able to enhance TGF-b-induced EMT in hepatocytes. We also analyzed the histological changes of the liver. As shown in Figure 5D and 5E, H&E staining and Oil-Red-O staining revealed that hepatic steatosis was induced by chronic alcohol exposure. Furthermore, the alcohol-induced liver steatosis was profoundly enhanced by Smad7 deletion. Consistently, Smad7 deletion led to a significant increase in the content of triglyceride level in the liver upon alcohol exposure. Together, these data suggest that the liver injury and steatosis induced by chronic alcohol administration were enhanced by Smad7 deletion, further indicating that Smad7 deletion has a deteriorating effect on liver functions. Recently, it was reported that over-activation of TGF-b signaling may enhance alcohol-mediated liver damage by reducing expression of alcohol dehydrogenase 1. In wild type mice, alcohol administration significantly increased the mRNA level of ADH1 in the liver. Interestingly, alcoholinduced ADH1 upregulation in the liver was slightly reduced in Smad7-deleted mice. To further confirm the effect of Smad7 deletion on ADH1 expression, we BI-D1870 S6 Kinase? inhibitor isolated primary hepatocytes from the wild type and Smad7liver-KO mice. The level of mRNA region corresponding to exon 4 of Smad7 gene was significantly reduced in Smad7-deleted hepatocytes, confirming that Smad7 was deleted in these cells. Alcohol treatment could significantly SCH727965 elevate Smad7 expression. Furthermore, alcohol administration could stimulate the expression of ADH1 in hepatocytes. However, the expression level of ADH1 was significantly reduced in Smad7-deleted hepatocytes under both basal and alcohol-treated conditions, further indicating that Smad7 deletion can reduce AHD1 expression in the liver. As Smad7 deletion is associated with activation of TGF-b signaling, our observation is also constant with the hypothesis that hyperactivity of TGF-b signaling aggravates alcohol-mediated liver injury through downregulation of ADH1.