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 7α-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. 1α, 25-Dihydroxy-vitamin D3 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 α in the human CYP7A1 promoter. Mammalian two-hybrid, coimmunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation assays show that ligand-activated VDR specifically interacts with hepatocyte nuclear factor 4α (HNF4α) to block HNF4α interaction with coactivators or to compete with HNF4α 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.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grants DK44442, DK58379].
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.025155.
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ABBREVIATIONS: CYP7A1, cholesterol 7α-hydroxylase; FXR, farnesoid X receptor; PXR, pregnane X receptor; VDR, vitamin D receptor; SHP, small heterodimer partner; FGF, fibroblast growth factor; LCA, lithocholic acid; 1α, 25-(OH)2-VD3,1α, 25-dihydroxy-vitamin D3; RXR, retinoid X receptor; CYP24A1, sterol 24-hydroxylase; HEK, human embryonic kidney; BARE, bile acid response element; mBARE, mutations in the bile acid response element; PCR, polymerase chain reaction; TK, thymidine kinase; SMRT, silencing mediator of retinoid and thyroid receptors; NCoR-1, nuclear receptor corepressor-1; SRC-1, steroid receptor coactivator-1; HNF4α, hepatocyte nuclear factor 4α; PGC-1α, peroxisome proliferators activator receptor γ coactivator 1α; EtOH, ethanol; UAS, upstream activating sequence; Q-PCR, quantitative real-time polymerase chain reaction; CYP27A1, sterol 27-hydroxylase; TNT, transcription and translation; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; GRIP-1, glucocorticoid receptor interacting protein-1; CoIP, coimmunoprecipitation; Ct, threshold cycle; Luc, luciferase; UBC, ubiquitin C.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. - Received October 14, 2008.
- Accepted December 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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