GCDCA down-regulates gene expression by increasing Sp1 binding to the NOS-3 promoter in an oxidative stress dependent manner.

During the course of cholestatic liver diseases, the toxic effect of bile acids accumulation has been related to the decreased expression of endothelial nitric oxide synthase (NOS-3) and cellular oxidative stress increase. In the present study, we have investigated the relationship between these two biological events. In the human hepatocarcinoma cell line HepG2, cytotoxic response to GCDCA was characterized by the reduced activity of the respiratory complexes II+III, the increased expression and activation of the transcription factor Sp1, and a higher binding capacity of this at positions -1386, -632 and -104 of the NOS-3 promoter (pNOS-3). This was associated with a decreased promoter activity and a consequent reduction of NOS-3 expression. The use of antioxidants in GCDCA-treated cells caused a lower activation of Sp1 and the recovery of the pNOS-3 activity and NOS-3 expression and activity. Similarly, the specific inhibition of Sp1 resulted in the improvement of NOS-3 expression. Both, antioxidant treatment and Sp1 inhibition were associated with the reduction of cell death-related parameters. Bile duct ligation in rats confirmed in vitro results concerning the activation of Sp1 and the reduction of NOS-3 expression. Our results provide direct evidence for the involvement of Sp1 in the regulation of NOS-3 expression during cholestasis. Thus, the identification of Sp1 as a potential negative regulator of NOS-3 expression represents a new mechanism by which the accumulation of bile acids causes a cytotoxic effect through the oxidative stress increase, and provides a new potential target in cholestatic liver diseases.