Mechanism of Vitamin D Receptor Inhibition of Cholesterol 7α-Hydroxylase Gene Transcription in Human Hepatocytes

Abstract

ABSTRACT:Lithocholic acid (LCA) is a potent endogenous vitamin D receptor (VDR) ligand. In cholestasis, LCA levels increase in the liver and intestine. The objective of this study is to test the hypothesis that VDR plays a role in inhibiting cholesterol 7␣-hydroxylase (CYP7A1) gene expression and bile acid synthesis in human hepatocytes. Immunoblot analysis has detected VDR proteins in the nucleus of the human hepatoma cell line HepG2 and human primary hepatocytes. 1␣, 25-Dihydroxy-vitamin D 3 or LCA acetate-activated VDR inhibited CYP7A1 mRNA expression and bile acid synthesis, whereas small interfering RNA to VDR completely abrogated VDR inhibition of CYP7A1 mRNA expression in HepG2 cells. Electrophoretic mobility shift assay and mutagenesis analyses have identified the negative VDR response elements that bind VDR/retinoid X receptor ␣ in the human CYP7A1 promoter. Mammalian twohybrid, coimmunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation assays show that ligandactivated VDR specifically interacts with hepatocyte nuclear factor 4␣ (HNF4␣) to block HNF4␣ interaction with coactivators or to compete with HNF4␣ for coactivators or to compete for binding to CYP7A1 chromatin, which results in the inhibition of CYP7A1 gene transcription. This study shows that VDR is expressed in human hepatocytes and may play a critical role in the inhibition of bile acid synthesis, thus protecting liver cells during cholestasis.

Establishment of a Stable Human Cell Line, HPL-A3, for Use in Reporter Gene Assays of Cytochrome P450 3A Inducers

“…Although the expression level of VDR is not especially high in human hepatocytes, 41,42) it has been reported that the activation of VDR led to modifications in the expression of hepatic CYPs, such as CYP3A4, 8) CYP7A1 and CYP24A1, 10) and to altered hepatic lipid metabolism. 43) The previous and present findings therefore suggest that HPL-A3 is a useful tool for the screening of hVDR activators/inhibitors and for the analysis of hVDR function in the liver.…”

“…6,7) Likewise, vitamin D receptor (VDR) 8) and constitutive androstane receptor (CAR), 9) which also act as a transcriptional activators of CYP3A subfamily genes, are expressed at low levels or not at all in human hepatoma-derived cell lines, including HepG2. 5,10) Recently, several stable cell lines for a human PXR (hPXR)-based reporter gene assay for the screening of CYP3A4 inducers have been established by co-transfection into human cell lines of an hPXR expression vector and a plasmid containing a reporter gene and hPXR-binding elements. [11][12][13][14][15] However, the available data on the xenobiotic-mediated induction of CYP3A enzymes at the levels of mRNA and activity in the reported cell lines are limited.…”

Retinoic Acid-related Orphan Receptor α Regulates Diurnal Rhythm and Fasting Induction of Sterol 12α-Hydroxylase in Bile Acid Synthesis

“…Expression plasmids for CBP and Gal4-CBP were gifts from Dr. Carolyn Smith (Baylor College of Medicine, Houston, TX). Plasmids for peroxisome proliferatoractivated receptor ␥ co-activator 1␣ (PGC1␣), Gal4-PGC1␣, Gal4-steroid receptor co-activator (SRC1), and Gal4-SRC2 were generated as reported previously (29).…”

“…All primers/probe sets for real-time PCR were ordered from TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). For assay of ROR␣ and GAPDH mRNA, SYBR Green primers (Applied Biosystems) were used as reported previously (29). Amplification of ubiquitin C and GAPDH were used as internal controls.…”

Bile acids: regulation of synthesis

“…The secondary bile acid LCA is a ligand of PXR and VDR. These two receptors bind to the BARE-I sequence in the human CYP7A1 promoter and inhibit CYP7A1 promoter activity ( 131,132 ). PXR interacts with HNF4 ␣ and blocks HNF4 ␣ recruitment of PGC-1 ␣ to CYP7A1 chromatin and results in inhibiting CYP7A1 transcription.…”