Nuclear Factor Erythroid 2-Related Factor 2 Is a Positive Regulator of Human Bile Salt Export Pump Expression

The bile salt export pump (BSEP) is the major determinant of bile salt-dependent bile secretion, and its deficiency leads to cholestatic liver injury. BSEP/Bsep gene expression is regulated by the nuclear farnesoid X receptor. However, BSEP expression, though reduced, is retained in the livers of Fxr(-/-) mice, indicating that additional transcriptional factors may regulate its expression. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a major role in response to oxidative stress by binding to antioxidant-responsive elements that regulate many hepatic phase I and 11 enzymes as well as hepatic efflux transporters. Computer software analysis of human BSEP reveals two musculo-aponeurotic fibrosacroma (Maf) recognition elements (MAREs) from the sequence in the proximal promoter region where Nrf2 may bind. In this study, we assessed whether Nrf2 plays a role in human BSEP expression and if this might be mediated by MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP messenger RNA expression by approximately seven-fold in HepG2 cells and protein by approximately 70% in human hepatocytes. Small interfering RNAs lowered NRF2 expression in HepG2 cells and prevented the up-regulation of BSEP by oltipraz. Human BSEP promoter activity was stimulated by Nrf2 in a dose-dependent manner in luciferase reporter assays. Mutations of the predicted MARE1, but not MARE2, abolished this Nrf2 transcriptional activation. Chromatin immunoprecipitation assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays further demonstrated direct binding of MARE1 in the BSEP promoter. Conclusion: Nrf2 is a positive transcriptional regulator of human BSEP expression. Pharmacological activation of Nrf2 may be beneficial for cholestatic liver injury. (HEPATOLOGY 2009;50:1588-1596.)