Iwayanagi Yuki, Takada Tappei, Suzuki Hiroshi
Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
Pharm Res. 2008 May;25(5):1134-41. doi: 10.1007/s11095-007-9496-9. Epub 2007 Dec 14.
Niemann-Pick C1-like 1 (NPC1L1) has been identified as a target of ezetimibe and found to be responsible for intestinal cholesterol absorption. Although, it was recently demonstrated that sterol responsive element binding protein 2 (SREBP2) is responsible for the cholesterol-dependent down-regulation of NPC1L1, the molecular mechanism of NPC1L1 expression is not fully understood. In the present study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4alpha), a key modulator of lipid metabolism, in the transcriptional regulation of human NPC1L1 gene.
Reporter gene assays and EMSAs were performed using human NPC1L1 promoter constructs and the effect of siHNF4alpha was examined.
Transfection of SREBP2 induced the transcriptional activities of NPC1L1 and additional transfection of HNF4alpha results in a marked stimulation of the activities. Studies with deletion mutants indicated that important elements are located within 264 nt upstream in the human NPC1L1 promoter. In addition, studies with mutations in putative binding sites of HNF4alpha indicated the existence of binding sites in -209 to -197 and -52 to -40. Moreover, HNF4alpha knockdown resulted in the reduced expression and regulation by cholesterol.
It is concluded that HNF4alpha plays a crucial role in the expression and regulation of human NPC1L1 gene.
尼曼-匹克C1样1蛋白（NPC1L1）已被确定为依泽替米贝的作用靶点，并被发现与肠道胆固醇吸收有关。尽管最近有研究表明，固醇调节元件结合蛋白2（SREBP2）负责胆固醇依赖性的NPC1L1下调，但NPC1L1表达的分子机制尚未完全阐明。在本研究中，我们检测了脂质代谢的关键调节因子肝细胞核因子4α（HNF4α）在人NPC1L1基因转录调控中的作用。
使用人NPC1L1启动子构建体进行报告基因检测和电泳迁移率变动分析（EMSA），并检测siHNF4α的作用。
转染SREBP2可诱导NPC1L1的转录活性，额外转染HNF4α可显著增强该活性。缺失突变体研究表明，重要元件位于人NPC1L1启动子上游264 nt范围内。此外，对HNF4α假定结合位点进行突变的研究表明，在-209至-197以及-52至-40存在结合位点。此外，敲低HNF4α会导致表达降低以及胆固醇对其调控作用减弱。
得出结论，HNF4α在人NPC1L1基因的表达和调控中起关键作用。
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