Mechanism of vitamin D receptor inhibition of cholesterol 7alpha-hydroxylase gene transcription in human hepatocytes.
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 7alpha-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. 1alpha, 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 alpha 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 4alpha (HNF4alpha) to block HNF4alpha interaction with coactivators or to compete with HNF4alpha 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.
Han
Shuxin
S
Department of Integrative Medical Sciences, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, 4209 State Route 44, Rootstown, OH 44272, USA.
Chiang
John Y L
JY
D001483:Base Sequence; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D055785:Gene Knockdown Techniques; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D047468:Immunoprecipitation; D008095:Lithocholic Acid / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol; D008095:Lithocholic Acid; D002790:Cholesterol 7-alpha-Hydroxylase