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Degree

PHD, Kent State University, College of Arts and Sciences / School of Biomedical Sciences, 2009.

Abstract

Vitamin D receptor (VDR) plays an important role in the regulation of calcium and phosphate homeostasis and bone formation. Lithocholic acid (LCA) is a potent endogenous ligand of VDR. 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 the gene expression of cholesterol 7α-hydroxylase (CYP7A1) and bile acid synthesis in human hepatocytes. Immunoblot analysis has detected VDR proteins in the human hepatoma cell line HepG2 and primary hepatocytes. 1α, 25-dihydroxy-vitamin D3 (1α,25(OH)2-VD3) or LCA acetate-activated VDR inhibited CYP7A1 mRNA expression and bile acid synthesis, whereas small interfering RNA (siRNA) to VDR completely abrogated VDR inhibition of CYP7A1 in HepG2 cells. LCA or 1α, 25(OH)2-VD3 induced VDR translocation from the cytosol to the nucleus and plasma membrane in human primary hepatocytes. LCA or 1α, 25(OH)2-VD3 activated a tyrosine kinase c-Src and the VDR signaling pathway, which activated c-Raf, an upstream kinase of the MAPK pathway. Kinase inhibition and in vitro kinase assays showed that VDR specifically activated extracellular signal-regulated kinase 1/2 (ERK1/2), which phosphorylated VDR, retinoid X receptor α (RXRα) and hepatocyte nuclear factor 4α (HNF4α). Mammalian two-hybrid, coimmunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation assays show that ligand activated VDR specifically interacts with HNF4α to block HNF4α interaction with coactivators. ERK1/2 inhibitors suppressed VDR interaction with HNF4α. Electrophoretic mobility shift assay and mutagenesis analyses have identified the negative VDR response elements that bind VDR/RXRα in the human CYP7A1 promoter. Chromatin immunoprecipitation assays show that LCA or 1α, 25(OH)2-VD3 activated ERK1/2 stimulated the recruitment of VDR, RXRα and co-repressors, but decreased the occupancy of HNF4α and coactivators to human CYP7A1 chromatin, resulting in the inhibition of CYP7A1 gene transcription. In conclusion, LCA and 1α, 25(OH)2-VD3 caused intracellular translocation of VDR to the plasma membrane and nucleus in human hepatocytes. LCA and VDR activated the c-Raf/MEK/ERK1/2 pathway to regulate VDR inhibition of CYP7A1 in hepatocytes. This study identified a novel bile acid-activated VDR signaling pathway in human hepatocytes. LCA activation of VDR may inhibit bile acid synthesis and protect hepatocytes from cholestatic liver injury.

Subject Headings

Biomedical research

Keywords

VDR; BILE; BILE ACID; CYP7A1; 1α; 2-VD3; ERK1/2

Advisor

John Chiang

Pages

255p.

Document number: kent1257459841
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=kent1257459841

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