Mechanism of Vitamin D Receptor Inhibition of Cholesterol 7α-Hydroxylase Gene Transcription in Human Hepatocytes
Mechanism of Vitamin D Receptor Inhibition of Cholesterol 7α-Hydroxylase Gene Transcription in Human Hepatocytes
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DOI：
10.1124/dmd.108.025155
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发表时间：
2009-03-01
影响因子：
3.9
通讯作者：
Chiang, John Y. L.
中科院分区：
文献类型：
--
作者：
Han, Shuxin;Chiang, John Y. L.
石胆酸(LCA)是一种有效的内源性维生素D受体(VDR)配体。在胆汁淤积症中，肝脏和肠道中的LCA水平增加。本研究的目的是验证维生素D受体在抑制人肝细胞胆固醇7α-羟基酶基因表达和胆汁酸合成方面的作用。免疫印迹分析在人肝癌细胞系HepG2和人原代肝细胞的细胞核中检测到VDR蛋白。1α，25-二羟基维生素D-3或LCA醋酸酯激活的VDR可抑制细胞色素P7A1基因的表达和胆汁酸的合成，而针对VDR的小干扰RNA可完全阻断VDR对细胞色素P7A1基因表达的抑制。电泳迁移率改变分析和突变分析已经确定了与人CYP7A1启动子中的VDR/维甲酸X受体α结合的VDR负反应元件。哺乳动物双杂交、免疫共沉淀、谷胱甘肽S转移酶下拉和染色质免疫共沉淀分析表明，配体激活的VDR与肝细胞核因子4α(HNF4α)特异性地相互作用，阻断HNF4α与共激活子的相互作用，或与HNF4α竞争共激活子，或竞争与细胞色素P7A1染色质的结合，从而抑制细胞色素P7A1基因的转录。本研究表明VDR在人肝细胞中表达，并可能在抑制胆汁酸合成中发挥重要作用，从而在胆汁淤积过程中保护肝细胞。
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 alpha-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 alpha, 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 4 alpha (HNF4 alpha) to block HNF4 alpha interaction with coactivators or to compete with HNF4 alpha 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.