Regulation of Bile Acid Synthesis by Nuclear Receptors in Vivo

胆汁酸是胆固醇和脂质的肠吸收和胆汁溶解所必需的。此外，本发明还提供了一种方法， 胆汁酸在胆固醇体内平衡中起关键作用。胆汁酸中有两种关键酶 生物合成途径，胆固醇7-羟化酶/CYP 7A 1（7-羟化酶），这是速率限制， “经典”途径的调节步骤，以及甾醇12-羟化酶/CYP 8B 1（12-羟化酶）， 胆酸合成的特异性酶。7-羟化酶和12-羟化酶基因的表达是高水平的。 受反馈调节机制的调节，主要在转录水平。的分子机制 参与这种调节似乎是相似的，并涉及法尼醇X的协调活动 受体（FXR）、小异源二聚体伴侣（SHP）和1-甲胎蛋白转录因子（FTF），也称为 如肝受体同源物-1（LRH-1）和肝细胞核因子-4（HNF-4）。迄今为止所做的所有研究 已经确定FTF是参与7-和12-羟化酶反馈调节的因子 已经在组织培养中进行了转录。鉴于它们DNA中核受体的混杂性 结合能力，确定FTF和HNF-4在体内胆汁酸生物合成中的作用至关重要。的 本提案的总体目标是了解FTF和HNF-4在反馈中的作用和调节 体内胆汁酸生物合成的调节。私家侦探假设胆汁酸介导了 通过两步机制调节7-和12-羟化酶的转录：首先， 胆汁酸通过p38 MAP激酶途径介导的过程抑制HNF-4（一种新的 最近在P.I.的实验室），并增加FTF的表达，从而导致FTF的增加。 与7-和12-羟化酶启动子中发现的重叠FTF/HNF-4识别位点结合 从该位点置换HNF-4，第二，FTF通过与SHP的异源二聚化而变得无活性。 初步研究表明，这些过程中涉及的一些机制是胆汁酸特异性的 并且其中一些机制在12-羟化酶的调节中发挥更大的作用。拟议 具体的研究包括：1）研究p38激酶在表达中的作用和作用机制， 2）阐明p38途径、FTF和HNF-4在7-和12-羟化酶基因中的作用， 胆汁酸介导的体内胆汁酸合成调节，利用具有抑制的p38活性的小鼠，和 FTF+/-和SHP-/-小鼠;和3）表征FTF从7-和12-小鼠中置换HNF-4， 羟化酶启动子，其参与胆汁酸介导的使用羟化酶的胆汁酸合成的调节。 相同的小鼠模型。这项研究的成功完成将为我们提供新的见解， 胆汁酸生物合成调控的分子机制。

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.