Transcriptional regulation of bile salt export pump

Bile salt export pump (BSEP) is the major bile salt transporter located in the canalicular membrane in the liver which controls the rate-limiting step in bile salt enterohepatic circulation and functions as the major driving force for bile formation. BSEP is regulated by the bile acid/farnesoid X receptor (FXR) signaling pathway. Metabolic conversion of cholesterol into bile acids in liver is initiated by the rate-limiting cholesterol 7α-hydroxylase (CYP7A1). Liver receptor homolog 1 (LRH-1) is a key transcriptional factor of CYP7A1. We investigated if LRH-1 was also involved in transcriptional regulation of BSEP and found that LRH-1 upregulated BSEP expression. A functional LRH-1 responsive element (LRHRE) was identified in the BSEP promoter by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. LRH-1 functioned as a modulator in bile acid/FXR-mediated BSEP regulation. LRH-1 was suggested to play a supporting role to FXR in maintaining hepatic bile acid levels by coordinately regulating CYP7A1 and BSEP for bile acid synthesis and elimination, respectively. An FXR response element (FXRE) with a characteristic inverted repeat, named IR1a, in the human BSEP promoter was previously reported. In the study, we identified a new FXRE, named IR1b, in the BSEP promoter and demonstrated that BSEP transactivation by FXR was mediated through the two distinct FXREs. Mutational studies revealed that that IR1b plays a predominant role in mediating the transactivation but IR1a is required for maximal activity. Sequence alignment revealed that IR1b was completely conserved among various species, whereas IR1a showed clear species differences. Substitution studies with IR1a from human, mouse and rat BSEP promoters demonstrated that IR1a was primarily responsible for the observed species difference in transactivation of BSEP by FXR. Malfunction of BSEP leads to a variety of cholestatic disorders. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy which is associated with sex hormone estrogens and BSEP. We hypothesized that estrogens down-regulate BSEP expression and such down-regulation is a risk factor for estrogens to induce intrahepatic cholestasis. In supporting our hypothesis, estrogen 17β-estradiol (E2) markedly decreased BSEP mRNA expression induced by bile acid in human hepatocytes and hepatoma Huh 7 cells. Estrogen receptor-alpha (ERα) dramatically repressed FXR-mediated BSEP promoter transactivation, and such repression was further enhanced by E2. ChIP assays demonstrated that ERα was specifically recruited to the FXR response elements (FXREs) in the BSEP promoter. Mutational analyses of ERα showed that ligand- but not DNA-binding was required for ERα to exert its repression on BSEP transactivation, indicating a non-classical estrogen/ERα pathway. Further studies with bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation assays revealed that ERα physically interacted with FXR. Disectional analysis of ERα function revealed that activation function -1 (AF1) domain is critical for repression of FXR transactivation. In conclusion, BSEP expression was down-regulated by estrogen through a non-classical pathway via the interaction between ERα and FXR. The finding potentially provides a molecular basis for developing new therapeutics to treat or prevent ICP through the intervention of the interaction between ERα and FXR