ChIP assays using Nrf2 antibody suggest there are AREs in the Mrp1-4 promoters(11). Nrf2 may bind. In this study we examine if Nrf2 plays a role in human BSEP expression and whether this might be mediated by the MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP mRNA expression by ~ 7-fold in HepG2 cells and protein by ~ 70% in human hepatocytes. siRNAs 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. ChIP assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays Myelin Basic Protein (87-99) further Elf3 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. Keywords:ATP-binding cassette (ABC) transporters, antioxidant-responsive element (ARE), oltipraz, bile secretion, gene regulation == INTRODUCTION == The bile salt export pump (BSEP, ABCB11) is a member of the ATP-binding cassette (ABC) superfamily of transporters(1). It is primarily expressed in the liver where it localizes to the canalicular membrane of hepatocytes. BSEP/Bsep is the major determinant of bile Myelin Basic Protein (87-99) salt-dependent bile secretion, and secretes monovalent conjugated bile acids from hepatocytes(2). Genetic deficiencies of BSEP lead to progressive cholestatic liver injury(3,4) and are risk factors for hepatocellular carcinoma(5). BSEP/Bsep expression is highly regulated by the nuclear farnesoid Myelin Basic Protein (87-99) X receptor (FXR, NR1H4) which heterodimerizes with the retinoid X receptor (RXR, NR2B), and binds to an inverted repeat (IR)-1 element in the BSEP promoter(6). BSEP/Bsep is also regulated by the liver receptor homolog-1 (LRH-1) and its expression is decreased in hepatocyte-specificLrh/mice(7). However, Bsep expression is still preserved in bothFxr/andLrh/mice(79), suggesting that there may be additional transcriptional factors that regulate its expression. The transcription factor NF-E2-related factor-2 (Nrf2) plays an important role in maintaining Redox homeostasis by regulating the expression of many Phase I and Phase II drug-metabolizing and detoxification enzymes, such as NAD(P)H quinone oxidoreductase (Nqo1), Myelin Basic Protein (87-99) and glutathione-S-transferase (GST)(10). In addition, Nrf2 also regulates a few Phase III transport proteins, including the multidrug resistance-associated proteins (Mrp) 14(11). Nrf2 is a 605 residue ubiquitous protein that belongs to the small family of basic leucine zipper transcription factors(12,13). Activation of Nrf2 is controlled by the actin-associated kelch-domain protein 1 (Keap1), which acts as a negative regulator of Nrf2(14). During basal conditions, Keap1 binds to Nrf2 and sequesters Nrf2 cytoplasmically(15). Under oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it heterodimerizes with the small musculo-aponeurotic fibrosacroma (Maf) protein. This complex then binds to an antioxidant responsive element (ARE) or an AP1-NF-E2 tandem repeat sequence, calledMafrecognitionelements (MARE), initiating transcription(12,16). Computer analysis reveals that human BSEP contains two MARE sites in its proximal promoter region, suggesting that Nrf2 may regulate BSEP expression. In the present study, we found that Nrf2 activators, in particular oltipraz (OPZ), induced BSEP mRNA and protein expression in HepG2 cells and human hepatocytes. Knockdown of Nrf2 by siRNA diminished the increase in BSEP mRNA expression. Human BSEP promoter reporter assays demonstrated that one of the computer-predicted MAREs mediated Nrf2 regulation in promoter activity. The involvement of Nrf2 in regulation of BSEP expression was further confirmed by ChIP and gel mobility shift assays in HepG2 cells. These findings indicate that Nrf2 is a positive regulator of BSEP expression, suggesting activation of Nrf2 might be of benefit in cholestatic liver injury. == EXPERIMENTAL PROCEDURES == == Chemical == Oltipraz was purchased from Axxora (San Diego, CA). Other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Polyclonal antibody against human BSEP was purchased from Kamiya Biomedical (Seattle, WA). Polyclonal antibody against human Nrf2 (sc-722) and siRNA-Nrf2 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). siRNA-control (a non-targeting siRNA) was purchased from Ambion (Foster, CA). A dual-luciferase assay kit was purchased from Promega (Madison, WI). == Cell cultures == The HepG2 cell line was obtained from the American Type Culture Collection (Manassas, VA) and cultured in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum (Invitrogen; Carlsbad, CA), 100 units/ml penicillin G, and 100 g/ml streptomycin. Primary human hepatocytes were obtained from the Liver Tissue Procurement and Distribution System of the National Institutes of Health (Dr. Stephen Strom; University of Pittsburgh, PA).