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: 6346126
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6346126
• 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.

在经典的反馈抑制环路中，肠肝中的胆汁酸 循环抑制限速酶的转录 胆汁酸生物合成途径胆固醇7α-羟化酶 (C7α H)。 已经提出了两个模型来解释这个问题 反馈抑制。 在类固醇激素型模型中，胆汁酸可能 与胞质受体结合，易位至细胞核，并相互作用 直接与 C7alphaH 启动子连接。 或者，胆汁酸可以 产生核外信号，导致修饰 C7alpha H 基因表达所需的转录因子。 数据 我们的实验室表明胆汁酸激活肝细胞 蛋白激酶 C (PKC)，并且 PKC 激活是 牛磺胆酸盐抑制 C7α H 转录。 具体的 目的：(1) 确定哪些 PKC 同工型调节 C7alphaH 转录； (2) 表征胆汁酸的作用机制 激活纯化的 PKC 同工型； (3) 比较佛波酯和 C7alphaH 5'侧翼区域胆汁酸诱导的事件；并且，(4) 探讨胆汁酸肠肝循环的影响 肝和回肠粘膜 PKC 同种型激活以及 这种激活对胆汁酸调节基因的可能影响。 实验设计：在培养的大鼠肝细胞（对象#1）中，Western 免疫印迹和RNA酶保护测定将用于 确定哪些 PKC 同种型易位至肝细胞 膜响应胆汁酸，并抑制 C7alpha H mRNA 水平。 在用杆状病毒重建的测定系统中- 表达 PKC 同工型（Obj. #2），我们将确定胆汁是否 酸与 PKC 同工型的调节域结合，或激活 通过促进它们与膜的结合来间接地作用它们。 在 转染的大鼠肝细胞（观察#4），RNase 保护测定， 免疫组织化学和原位杂交将用于 记录胆汁酸诱导的 PKC 亚型的重要性 激活调节肝窦胆汁酸 C7alphaH 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通量 可以动态调节肝脏和肝脏的活动和周转 肠道 PKC 亚型，并且 PKC 亚型依次协调 胆汁酸转运蛋白和生物合成酶的活性。