MOLECULAR REGULATION OF SMOOTH MUSCLE DIFFERENTIATION

平滑肌分化的分子调控

• 批准号: 2735130
• 负责人: Gary K Owens
• 金额: $ 25.05万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2735130
• 关键词: DNA footprinting; actins; antisense nucleic acid; aorta; atherosclerosis; cell differentiation; cell growth regulation; developmental genetics; gel mobility shift assay; genetic promoter element; genetic regulatory element; genetically modified animals; laboratory mouse; laboratory rat; muscle cells; myogenesis; nucleic acid sequence; site directed mutagenesis; tissue /cell culture; transcription factor; vascular smooth muscle

DESCRIPTION (Adapted from Investigator's Abstract): Alterations in the differentiated state of the smooth muscle cell (SMC) are believed to play a key role in the development and/or progression of atherosclerotic disease. The long-term goal of this proposal is to identify molecular mechanisms that control differentiation/maturation of SMC through elucidation of mechanisms that control transcription of SM alpha-actin, a contractile protein that is selective for SMC and which is required for its major differentiated function, contraction. Aim 1 of this proposal is to identify cellular and molecular mechanisms that regulate cell-type specific expression of the SM alpha-actin gene both in cultured SMC and in vivo in transgenic animals. Studies will include: a) further characterization of DNA sequences required for SMC specific transcriptional regulation via two highly conserved CC(AT)6GG (CarG) elements that the applicant has previously demonstrated are required for high transcriptional activity in SMC; b) identification of trans factors that interact with and regulate the activity of these CarG elements; c) determination of the minimal promoter sequences required for expression of the SM alpha-actin gene in transgenic animals; d) identification of cis elements responsible for the positive transcriptional activity of the first intron observed in our preliminary studies; e) identification of cis elements upstream of the core 125 bp SM alpha-actin promoter that are required for cell-type-specific expression of this gene; and f) determination if the positive activity of the E-boxes of the SM alpha-actin promoter are mediated by a novel SMC specific HLH transcription factor. Aim 2 will be to determine the in vivo function of selected cis elements and trans factors identified in Aim 1. Studies will include testing the activity of selected cis elements in transgenics by site-directed mutagenesis, and determining the temporal pattern of expression of SM alpha-actin transcription factors relative to alterations in expression of SM alpha-actin and other SMC differentiation marker proteins during vasculogenesis, and following vascular injury. Aim 3 will be to determine whether there is evidence for coordinate regulation of transcription of SM alpha-actin and genes encoding other SMC differentiation marker proteins, and to determine the underlying mechanisms. Studies represent an extension of ongoing work, and will contribute to understanding of mechanisms that contribute to alterations in SMC differentiation control processes in vascular disease. In addition, studies are likely to identify DNA regulatory sequences that confer SMC specific expression which could be used for construction of vectors for SMC specific gene knockouts and/or targeting gene therapies to the vasculature.

描述（根据研究者的摘要进行了改编）： 平滑肌细胞（SMC）的差异化状态可以发挥 动脉粥样硬化疾病的发展和/或进展中的关键作用。 该提议的长期目标是确定分子机制 通过阐明机制来控制SMC的分化/成熟 SMα-肌动蛋白的控制转录，一种收缩蛋白，是 SMC的选择性，这是其主要差异化所必需的 功能，收缩。 该建议的目标1是识别细胞和 调节SM的细胞类型特异性表达的分子机制 转基因动物中培养的SMC和体内α-肌动蛋白基因。 研究将包括：a）需要进一步表征DNA序列所需的DNA序列 通过两个高度保守的SMC特异性转录调节 申请人以前证明的CC（AT）6GG（CARG）元素是 SMC中高转录活性所必需的； b）识别 与这些CARG相互作用并调节活性相互作用并调节的反式因子 元素； c）确定最小启动子序列 转基因动物中SMα-肌动蛋白基因的表达； d） 识别负责阳性转录的顺式元件 在我们的初步研究中观察到的第一个内含子的活性； e） 核心125 bp smα-肌动蛋白上游的顺式元件的识别 该基因的细胞类型表达所需的启动子； f）确定SM的电子盒的正活性是否 α-肌动蛋白启动子由新型的SMC特异性HLH转录介导 因素。 AIM 2将是确定所选顺式的体内功能 AIM 1中确定的元素和反式因素。研究将包括 测试通过 定向诱变，并确定 SMα-肌动蛋白转录因子相对于改变的表达 在SMα-肌动蛋白和其他SMC分化标记的表达中 血管生成期间的蛋白质以及血管损伤后。 目标3意志 确定是否有证据表明对 SMα-肌动蛋白和编码其他SMC分化的基因的转录 标记蛋白，并确定基本机制。 研究 代表正在进行的工作的扩展，并将有助于理解 有助于改变SMC分化控制的机制 血管疾病的过程。 此外，研究可能会确定 DNA调节序列可以赋予SMC特定表达的DNA调节序列 用于构建用于SMC特定基因敲除和/或的向量的载体 针对脉管系统的基因疗法。