REGULATION OF CHOLESTEROL ESTERASE IN STEROIDOGENESIS

胆固醇酯酶在类固醇生成中的调节

• 批准号: 2713403
• 负责人: FREDRIC B. KRAEMER
• 金额: $ 15.7万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2713403
• 关键词: RNase protection assay; adrenal glands; adrenocorticotropic hormone; antisense nucleic acid; cholesterol esters; chorionic gonadotropin; enzyme activity; enzyme biosynthesis; enzyme induction /repression; enzyme mechanism; enzyme structure; estradiol; genetic transcription; gonadotropins; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; laboratory rat; messenger RNA; ovary; phosphorylation; steroid biosynthesis; sterol esterase; tissue /cell culture; transfection

The presence of substantial pools of cholesteryl esters in the adrenal cortex and ovary, and the recognition that this potential substrate enters the steroidogenic pathway as free cholesterol, signifies the important role of cholesterol esterase in the regulation of steroidogenesis. The neutral cholesteryl ester hydrolase (CEH) activity purified from the adrenal and the ovary appears to be identical to hormone-sensitive lipase (HSL) purified from adipose tissue. While significant information has been generated to advance the understanding of some of the mechanisms acutely regulating HSL activity in adipose tissue, very little data exist concerning changes in the expression of CEH and the factors and mechanisms regulating its expression in steroidogenic tissues. Similarly, the consequences of the action of HSL as a neutral cholesterol esterase have not been directly established in regard to influencing steroidogenesis. Moreover, the regulation of neutral CEH activity has been shown to be rapidly modulated by hormones via phosphorylation-dephosphorylation reactions, yet, very little information exists on the mechanisms involved in regulating HSL other than posttranslational modification. To address these issues, the overall goals of this proposal are to understand the mechanisms regulating the expression of neutral CEH in steroid-producing cells and to establish the structure-function relationships of HSL as a neutral cholesterol esterase. The first aim is to explore the mechanisms regulating the physiological expression of neutral CEH in steroidogenic cells. The mechanisms regulating neutral CEH in vivo and in vitro in steroidogenic cells will be examined by following changes in CEH activity, the amount of HSL protein, the steady-state mRNA levels, and the rate of transcription of HSL mRNA in the adrenal and ovary during relevant hormonal manipulations. In addition, the mechanisms responsible for activation of HSL-mediated cholesteryl ester hydrolysis by trophic hormones will be explored by examining the possible translocation of HSL. The second aim is to establish the structure-function relationships of HSL as a neutral cholesteryl esterase in steroidogenic cells and its subsequent effects on steroidogenesis. Murine adrenal cell lines and transformed rat granulosa cells will be stably transfected, while primary cultures of adrenal, granulosa, and luteal cells will be transiently transfected, to overexpress normal HSL and the ability of the cells to accumulate cholesteryl esters and to produce steroids will be examined. CHO, adrenal, and granulosa cell lines will be transfected with mutated forms of rat HSL to examine the function of HSL and the structural relationships of phosphorylation and the putative lipid binding domain. Finally, steroidogenic cell lines will be stably transfected with an antisense hammerhead ribozyme construct, or antisense oligonucleotides will be used in primary cultures of adrenal, granulosa and luteal cells, to eliminate HSL expression to examine the importance of HSL in cholesteryl ester hydrolysis and its actions on steroidogenesis. The results of this proposal will delineate the function of HSL, the mechanisms controlling its expression, and its contribution to the regulation of steroidogenesis-linked cholesterol ester hydrolysis.

肾上腺中存在大量胆汁胆料酯的存在 皮层和卵巢，以及这种潜在底物进入的认识 类固醇生成途径作为游离胆固醇，表示重要 胆固醇酯酶在类固醇发生的调节中的作用。这 中性胆固醇酯水解酶（CEH）活性从纯净的 肾上腺和卵巢似乎与激素敏感脂肪酶相同 （HSL）从脂肪组织中纯化。虽然重要的信息已经 生成以促进对某些机制的理解 调节脂肪组织中的HSL活性，存在很少的数据 关于CEH表达的变化以及因素和机制 调节其在类固醇组织中的表达。同样， HSL作为中性胆固醇酯酶的作用的后果具有 没有直接建立在影响类固醇生成方面。 此外，已证明中性CEH活性的调节是 通过磷酸化 - 二磷酸化迅速通过激素调节 反应，但关于涉及机制的信息很少 在调节翻译后修饰以外的其他HSL方面。解决 这些问题，该提案的总体目标是了解 调节类固醇生产中中性CEH表达的机制 细胞并建立HSL的结构功能关系 中性胆固醇酯酶。第一个目的是探索机制 调节类固醇中性CEH的生理表达 细胞。调节体内中性CEH和体外的机制 类固醇生成细胞将通过以下CEH活性的变化来检查 HSL蛋白的量，稳态mRNA水平和速率 相关期间，肾上腺和卵巢中HSL mRNA的转录 荷尔蒙操纵。另外，负责的机制 hsl介导的胆固醇酯的激活通过营养 通过检查HSL的可能易位，将探索激素。 第二个目的是建立HSL的结构功能关系 作为类固醇生成细胞中的中性胆固醇酯酶及其 随后对类固醇生成的影响。鼠肾上腺细胞系和 转化后的大鼠颗粒细胞将稳定转染，而主要的 肾上腺，颗粒和黄体细胞的培养物将是瞬时的 转染，过表达正常HSL和细胞的能力 将检查积累胆固醇酯并产生类固醇。 CHO，肾上腺和颗粒细胞系将用突变转染 大鼠HSL的形式检查HSL和结构的功能 磷酸化和推定的脂质结合结构域的关系。 最后，类固醇生成细胞系将稳定转染 反义锤头核酶构建体或反义寡核苷酸 将用于肾上腺，颗粒和黄体细胞的原发性培养物， 消除HSL表达以检查HSL在 胆固醇酯的水解及其对类固醇生成的作用。这 该提案的结果将描述HSL的功能 控制其表达的机制及其对 调节类固醇相关的胆固醇酯水解。