REGULATION OF BILE ACID SYNTHESIS--CHOLESTEROL 7ALPHA HYDROXYLASE

胆汁酸合成的调控--胆固醇7α羟化酶

• 批准号: 6201845
• 负责人: Z. R VLAHCEVIC
• 金额: $ 14.86万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6201845
• 关键词: DNA footprinting; RNase protection assay; binding proteins; cholanate compound; enzyme activity; enzyme induction /repression; gel mobility shift assay; genetic transcription; immunocytochemistry; in situ hybridization; laboratory rat; liver cells; liver circulation; messenger RNA; molecular cloning; northern blottings; nucleic acid sequence; phorbols; polymerase chain reaction; protein isoforms; protein kinase C; steroid 7alpha hydroxylase; steroid biosynthesis; tissue /cell culture; western blottings

In a classical feedback inhibitory loop, bile acids in the enterohepatic circulation repress the transcription of the rate-limiting enzyme of the bile acid biosynthetic pathway cholesterol 7 alpha-hydroxylase (C7alpha H). Two models have been proposed to account for this feedback inhibition. In a steroid hormone-type model, bile acids may bind to a cytosolic receptor, translocate to the nucleus, and interact directly with the C7alphaH promoter. Alternatively, bile acids may generate extranuclear signals, resulting in modification of a transcription factor required for C7alpha H gene expression. Data from our laboratories suggest that bile acids activate hepatocellular protein kinase C (PKC), and that PKC activation is required for the repression of C7alpha H transcription by taurocholate. Specific Aims: (1) To determine which PKC isoforms regulate C7alphaH transcription; (2) to characterize the mechanisms by which bile acids activate purified PKC isoforms; (3) to compare phorbol ester- and bile acid-induced events at the C7alphaH 5'-flanking region; and, (4) to explore the effects of the enterohepatic circulation of bile acids on hepatic and ileal mucosal PKC isoform activation as well as the possible implications of this activation on bile acid-regulated genes. Experimental design: In cultured rat hepatocytes (Obj. #1), Western immunoblotting and RNASE protection assays will be used to determine which PKC isoforms are translocated to hepatocellular membranes in response to bile acids, and which repress C7alpha H mRNA levels. In reconstituted assay systems with baculovirus- expressed PKC isoforms (Obj. #2), we will determine whether bile acids bind to the regulatory domain of PKC isoforms, or activate them indirectly by facilitating their association with membranes. In transfected rat hepatocytes (Obj. #4), RNase protection assays, immunohistochemistry, and in situ hybridization will be used to document the significance of bile acid-induced PKC isoform activation in regulating C7alphaH, the hepatic sinusoidal bile acid transporter, and the ileal bile acid transporter. Significance: We postulate that the flux of bile acids in the enterophepatic circulation may dynamically regulate the activity and turnover of hepatic and intestinal PKC isoforms, and that PKC isoforms in turn coordinate the activity of bile acid transporters and biosynthetic enzymes.

在一个经典的反馈抑制环中，胆汁酸在肠肝 循环抑制细胞中限速酶的转录 胆汁酸生物合成途径胆固醇7α-羟基酶 (C7Alpha H)。已经提出了两个模型来解释这一点 反馈抑制。在类固醇激素类型的模型中，胆汁酸可能 与胞质受体结合，移位到细胞核，并相互作用 直接与C7alphaH启动子结合。或者，胆汁酸可以 产生核外信号，导致一种 C7alphaH基因表达所需的转录因子。数据 来自我们实验室的研究表明，胆汁酸可以激活肝细胞 蛋白激酶C(PKC)，而PKC的激活是 牛磺胆酸盐抑制C7αH转录。特定的 目的：(1)确定哪些PKC亚型调节C7alphaH 转录；(2)描述胆汁酸的作用机制 激活纯化的PKC亚型；(3)比较佛波酯和佛波酯 胆汁酸诱导的C7alphaH5‘侧翼区事件；以及，(4) 探讨胆汁酸的肠-肝循环对大鼠心脏功能的影响。 肝和回肠粘膜PKC亚型的激活以及 这种激活对胆汁酸调节基因的可能影响。 实验设计：培养大鼠肝细胞(Obj.#1)，西部 免疫印迹和核糖核酸酶保护分析将用于 确定哪些PKC亚型转位到肝细胞 对胆汁酸作出反应并抑制C7alphaH的膜 M RNA水平。在重组的杆状病毒检测系统中- 表达的PKC亚型(Obj.#2)，我们将确定胆汁是否 酸与PKC异构体的调节域结合，或激活 通过促进它们与膜的结合，间接地促进它们的存在。在……里面 转基因大鼠肝细胞(Obj.#4)，核糖核酸酶保护分析， 免疫组织化学和原位杂交将用于 胆汁酸诱导的蛋白激酶C亚型的意义 激活对肝窦胆汁酸C7AlphaH的调节作用 转运蛋白和回肠胆汁酸转运蛋白。意义：我们 假设胆汁酸在肠肝循环中的流量 可以动态调节肝脏和肝脏的活动和周转。 肠道PKC异构体，PKC异构体依次协调 胆汁酸转运体和生物合成酶的活性。