Department of Pharmacy, The University of Tokyo Hospital Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.
Pharm Res. 2011 Feb;28(2):405-12. doi: 10.1007/s11095-010-0294-4. Epub 2010 Oct 16.
Niemann-Pick C1-like 1 (NPC1L1), a pharmacological target of ezetimibe, is responsible for cholesterol absorption in enterocytes and hepatocytes. In the present study, the involvement of peroxisome proliferator-activated receptor α (PPARα) and its cofactor, PPARγ coactivator 1α (PGC1α) in the transcriptional regulation of human NPC1L1 was analyzed.
Reporter gene assays and electrophoretic mobility shift assays (EMSAs) were performed with the 5'-flanking region of the human NPC1L1 gene and the effect of siPPARα was examined.
PPARα-mediated transactivation was observed with human NPC1L1 promoter constructs. Detailed analyses using deletion- and mutated-promoter constructs revealed the presence of a functional PPARα-response element (PPRE) upstream of the human NPC1L1 gene (-846/-834), a direct binding of PPARα and RXRα to which was confirmed by EMSAs. Moreover, PPARα-specific knockdown resulted in a significant decrease in the endogenous expression of NPC1L1 mRNA and protein in human-derived HepG2 cells. Furthermore, cotransfection of PGC1α stimulated the SREBP2/HNF4α- and PPARα/RXRα-mediated activation of the human NPC1L1 promoter.
We found that PPARα positively regulates human NPC1L1 transcription via direct binding to a PPRE. Additionally, PGC1α stimulates the SREBP2/HNF4α- and PPARα/RXRα-mediated transactivation of human NPC1L1. These findings may provide new insights into the close relationship of glucose, fatty acids and cholesterol homeostasis.
尼曼-匹克 C1 样 1（NPC1L1）是依折麦布的药理靶点，负责肠细胞和肝细胞中的胆固醇吸收。在本研究中，分析了过氧化物酶体增殖物激活受体α（PPARα）及其共激活因子过氧化物酶体增殖物激活受体γ共激活因子 1α（PGC1α）在人 NPC1L1 转录调控中的作用。
采用人 NPC1L1 基因 5'侧翼区进行报告基因检测和电泳迁移率变动分析（EMSA），并检测 siPPARα 的作用。
观察到 PPARα 介导的人 NPC1L1 启动子构建体的转录激活。使用缺失和突变启动子构建体的详细分析显示，人 NPC1L1 基因上游存在功能性 PPARα 反应元件（PPRE）（-846/-834），通过 EMSA 证实了 PPARα 和 RXRα 的直接结合。此外，PPARα 特异性敲低导致人源性 HepG2 细胞中 NPC1L1 mRNA 和蛋白的内源性表达显著下降。此外，PGC1α 的共转染刺激了 SREBP2/HNF4α 和 PPARα/RXRα 介导的人 NPC1L1 启动子的激活。
我们发现 PPARα 通过直接结合 PPRE 正向调节人 NPC1L1 转录。此外，PGC1α 刺激 SREBP2/HNF4α 和 PPARα/RXRα 介导的人 NPC1L1 转录激活。这些发现可能为葡萄糖、脂肪酸和胆固醇稳态的密切关系提供新的见解。
