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.

在经典的反馈抑制环中，肠肝中的胆汁酸 循环抑制速率限制酶的转录 胆汁酸生物合成途径胆固醇7α-羟化酶 （C7alpha H）。 已经提出了两种模型来解决这个问题 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，转移到核并相互作用 直接与C7alphah启动子一起。 或者，胆汁酸可能 生成核外信号，导致修改 C7alpha H基因表达所需的转录因子。 数据 从我们的实验室提出，胆汁酸会激活肝细胞 蛋白激酶C（PKC），并且需要PKC激活 通过牛旋酸酯对C7alpha H转录的抑制。 具体的 目的：（1）确定哪种PKC同工型调节C7alphah 转录； （2）表征胆汁酸的机制 激活纯化的PKC同工型； （3）比较凤凰酯和 胆汁酸诱导的C7alphah 5'-边缘区域的事件；和（4） 探索胆汁酸的肠肝循环的影响 肝和回肠粘膜PKC同工型激活以及 这种激活对胆汁酸调节的基因的可能影响。 实验设计：在培养的大鼠肝细胞（OBJ。＃1）中，西方 免疫印迹和RNase保护分析将用于 确定哪些PKC同工型易位到肝细胞 膜响应胆汁酸，并抑制c7alpha h mRNA水平。 在重构的测定系统中 表达的PKC同工型（OBJ。＃2），我们将确定是否胆汁 酸与PKC同工型的调节结构域结合或激活 它们通过促进与膜的关联间接地。 在 转染的大鼠肝细胞（OBJ。＃4），RNase保护分析， 免疫组织化学和原位杂交将用于 记录胆汁酸诱导的PKC同工型的意义 在调节C7Alphah的激活，肝弦胆汁酸 转运蛋白和回肠胆汁酸转运蛋白。 意义：我们 假设肠ep虫循环中的胆汁酸的通量 可能会动态调节肝的活动和离职 肠道PKC同工型，以及PKC同工型依次坐标 胆汁酸转运蛋白和生物合成酶的活性。