Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7α-hydroxylase gene transcription

Abstract

Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7α-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4α (HNF4α, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4α. PXR also interacted with peroxisome proliferator-activated receptor γ coactivator (PGC-1α), which interacted with HNF4α and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4α and reduced PGC-1α interaction with HNF4α. Chromatin immunoprecipitation assay showed that PXR, HNF4α, and PGC-1α bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1α from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4α and blocks PGC-1α activation with HNF4α and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis.