Regulation of Bile Acid Synthesis by Nuclear Receptors in Vivo

说明(申请人提供)：胆汁酸是肠道吸收和胆汁溶解胆固醇和脂肪所必需的。此外，胆汁酸在胆固醇动态平衡中起着至关重要的作用。胆汁酸生物合成途径中有两个关键酶：胆固醇7-羟基酶/CYP7A1(7-羟基酶)和固醇12-羟基酶/CYP8B1(12-羟基酶)，前者是经典途径的限速和调节步骤，后者是胆酸合成的特异性酶。7-和12-羟基酶基因的表达受反馈调控机制的高度调控，主要是在转录水平上。参与这一调控的分子机制似乎是相似的，涉及法尼类X受体(FXR)、小异二聚体(SHP)和1-甲胎蛋白转录因子(FTF)(又称肝脏受体同源1(LRH-1))和肝细胞核因子-4(HNF-4)的协调活性。到目前为止，所有确认FTF是参与7-和12-羟基酶转录反馈调节的因素的研究都是在组织培养中进行的。鉴于核受体DNA结合能力的混杂，确定FTF和HNF-4在体内胆汁酸生物合成中的作用是至关重要的。这项建议的总体目标是了解FTF和HNF-4在体内胆汁酸生物合成的反馈调节中的作用和调节。P.I.假设胆汁酸通过两步机制介导7-和12-羟基酶转录下调：首先，胆汁酸通过p38 MAP激酶途径抑制hnf-4(这是P.I.S实验室最近发现的一项新观察)，并增加FTF的表达，从而增加FTF与重叠的ftf/hnf-4识别位点的结合，这是在7-和12-羟基酶启动子中发现的取代hnF-4的位置，第二，FTF通过与SHP的异位二聚化而变得无效。初步研究表明，这些过程中涉及的一些机制是胆汁酸特异的，其中一些机制在12-羟基酶的调节中发挥更大的作用。拟进行的具体研究包括：1)研究p38激酶在7-和12-羟基酶基因表达中的作用和作用机制；2)利用p38活性抑制的小鼠、FTF和SHP-/-小鼠，阐明p38途径、FTF和HNF-4在胆汁酸介导的体内胆汁酸合成调节中的作用；3)利用相同的小鼠模型，研究FTF从7-和12-羟基酶启动子中取代HNF-4的作用，这参与了胆汁酸介导的胆汁酸合成的调节。这项研究的成功完成将为我们对胆汁酸生物合成调控的分子机制提供新的见解。与公共健康相关：胆汁酸是肠道吸收和胆汁溶解胆固醇和脂肪所必需的。此外，胆汁酸在胆固醇动态平衡中起着至关重要的作用。我们建议研究胆汁酸生物合成途径的新调节模式。这项研究的成功完成将为我们对胆汁酸生物合成调控的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Bile acids are required for intestinal absorption and biliary solubilization of cholesterol and lipids. In addition, bile acids play a crucial role in cholesterol homeostasis. There are two key enzymes in the bile acid biosynthetic pathways, cholesterol 7-hydroxylase/CYP7A1 (7-hydroxylase), which is the rate limiting and regulatory step of the "classic" pathway, and sterol 12-hydroxylase/CYP8B1 (12-hydroxylase), which is the specific enzyme for cholic acid synthesis. Expression of the 7- and 12-hydroxylase genes is highly regulated by feedback regulatory mechanisms, mainly at the transcriptional level. The molecular mechanisms involved in this regulation appear to be similar, and involve the coordinated activity of the farnesoid X receptor (FXR), the small heterodimer partner (SHP) and 1-fetoprotein transcription factor (FTF), also known as liver receptor homolog-1 (LRH-1), and hepatocyte nuclear factor-4 (HNF-4). All the studies done to date that have identified FTF as a factor involved in the feedback regulation of 7- and 12-hydroxylase transcription have been performed in tissue culture. Given the promiscuity of nuclear receptors in their DNA binding abilities, it is crucial to determine the role of FTF and HNF-4 in bile acid biosynthesis in vivo. The overall goal of this proposal is to understand the role and regulation of FTF and HNF-4 in the feedback regulation of bile acid biosynthesis in vivo. The P.I. hypothesizes that bile acids mediate the down regulation of the transcription of the 7- and 12-hydroxylases, by a two-step mechanism: first, bile acids suppress HNF-4 through a process mediated by the p38 MAP kinase pathway (a novel observation recently made in the P.I.'s lab), and increase FTF expression, which results in an increase in FTF binding to the overlapping FTF/HNF-4 recognition site found in the 7- and 12-hydroxylase promoters displacing HNF-4 from that site, and second, FTF becomes inactive through its heterodimerization with SHP. Preliminary studies suggest that some of the mechanisms involved in these processes are bile acid-specific and some of these mechanisms play a greater role in the regulation of 12-hydroxylase. The proposed specific studies are: 1) to characterize the role and mechanism of action of p38 kinase in the expression of the 7- and 12-hydroxylase genes; 2) to elucidate the role of the p38 pathway, FTF and HNF-4 in the bile acid-mediated regulation of bile acid synthesis in vivo, utilizing mice with suppressed p38 activity, and FTF and SHP-/- mice; and 3) to characterize the displacement of HNF-4 by FTF from the 7- and 12- hydroxylase promoters, that is involved in the bile acid-mediated regulation of bile acid synthesis using the same mouse models. The successful completion of this study will provide us with new insights into the molecular mechanisms involved in the regulation of bile acid biosynthesis. PUBLIC HEALTH RELEVANCE: Bile acids are required for intestinal absorption and biliary solubilization of cholesterol and lipids. In addition, bile acids play a crucial role in cholesterol homeostasis. We propose to study novel modes of regulation of the bile acid biosynthetic pathway. The successful completion of this study will provide us with new insights into the molecular mechanisms involved in the regulation of bile acid biosynthesis.