A1-ADRENERGIC REGULATION OF CARDIAC GENE EXPRESSION

心脏基因表达的 A1-肾上腺素调节

• 批准号: 2220318
• 负责人: PAUL C SIMPSON
• 金额: $ 25.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2220318
• 关键词: DNA directed RNA polymerase; alpha adrenergic receptor; binding proteins; disease /disorder model; gene expression; genetic regulation; genetic transcription; hormone receptor; laboratory rat; molecular pathology; newborn animals; norepinephrine; tissue /cell culture; transfection; ventricular hypertrophy

This work is focused on the clinical problem of myocardial hypertrophy, specifically the molecular mechanisms which regulate this process. A new model system was developed to study hypertrophy, employing neonatal heart myocytes in serum-free culture. The novel observation was made that stimulation of the alpha 1-adrenergic receptor on these cells by the catecholamine norepinephrine induces cell enlargement or hypertrophy, without DNA synthesis. The alpha 1-adrenergic receptor is the first receptor shown to regulate cardiac myocyte hypertrophy. Recent work has shown that the alpha 1 receptor regulates mRNA expression during hypertrophy. In particular, alpha 1 stimulation induces two contractile protein iso-mRNAs characteristic of early cardiac development and pressure-load hypertrophy in vivo, skeletal alpha-actin iso-mRNA and B-myosin heavy chain iso-mRNA. This alpha 1 receptor effect is selective and is mediated at the level of iso-gene transcription. Thus, the critical question is the mechanism for induction of transcription by the alpha 1 receptor. The present experiments will test the hypothesis that the skeletal alpha- actin iso-gene and the B-myosin heavy chain iso-gene contain one or more alpha 1 response elements, i.e., DNA sequences required specifically for transcription of these genes in response to alpha 1-adrenergic stimulation. Two complementary experimental approaches for functional identification alpha 1 response elements are proposed: (1) transfection of hybrid genes in a transient assay system; and (2) DNase I hypersensitivity mapping. The necessary cloned genes are available to begin these experiments and preliminary studies provide evidence that the work is feasible. The long-range goal is to define the intracellular pathway from the alpha 1 receptor at the cell surface to gene transcription. The overall hypothesis is that receptor stimulation activates a pre-existing protein or proteins that binds to the alpha 1 response element in a transcriptional complex with RNA polymerase II. Identification of an alpha 1 response element or elements in the present work will provide the foundation for subsequent identification of the protein or proteins.

这项工作集中在心肌肥厚的临床问题上， 具体地说，就是调节这一过程的分子机制。一种新的 利用新生儿心脏，开发了研究肥大的模型系统 无血清培养中的心肌细胞。这一新奇的观察结果是 对这些细胞上α1-肾上腺素能受体的刺激作用 儿茶酚胺去甲肾上腺素诱导细胞增大或肥大， 没有DNA合成。α1-肾上腺素能受体是第一个 受体具有调节心肌细胞肥大的作用。 最近的研究表明，阿尔法1受体调节信使核糖核酸的表达 在肥大期间。特别是，阿尔法1刺激诱导了两个 心脏早期发育所特有的收缩蛋白等位基因 和体内压力负荷性肥大，骨骼α-肌动蛋白等位基因和 B-肌球蛋白重链等位基因。这种阿尔法1受体效应是选择性的。 并在等基因转录水平上进行调节。因此， 关键问题是基因转录的诱导机制 α1受体。 目前的实验将检验这样的假设，即骨骼α- 肌动蛋白同源基因和B-肌球蛋白重链同源基因包含一个或多个 Alpha 1反应元件，即特定于 这些基因转录对α1-肾上腺素能的反应 刺激。 两种互补的功能鉴定实验方法 提出了Alpha-1反应元件：(1)杂交基因的导入 以及(2)DNase I超敏图谱。 必要的克隆基因可用于开始这些实验和 初步研究表明，这项工作是可行的。 长期目标是从阿尔法定义细胞内途径 1受体在细胞表面对基因转录起作用。整体而言 假设是受体刺激激活了一种预先存在的蛋白质。 或与α1反应元件结合的蛋白质 RNA聚合酶转录复合体的研究--II.An的鉴定 本工作中的一个或多个阿尔法1响应要素将提供 为随后鉴定一个或多个蛋白质奠定基础。