Transcriptional Regulation of PDGF Receptor Alpha

PDGF 受体 Alpha 的转录调控

• 批准号: 6779524
• 负责人: JANE E REUSCH
• 金额: $ 29.48万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-15 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779524
• 关键词: DNA footprinting; affinity chromatography; apolipoprotein E; atherosclerosis; cAMP response element binding protein; cell migration; cell proliferation; chromatin immunoprecipitation; diabetes mellitus; enhancer binding protein; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetically modified animals; growth factor receptors; laboratory mouse; mitogens; muscle cells; peroxisome proliferator activated receptor; platelet derived growth factor; protein binding; receptor expression; site directed mutagenesis; vascular smooth muscle

DESCRIPTION (provided by applicant): The long-term objective of our laboratory is to understand changes in gene expression and regulation that occur during insulin resistance and diabetes which permit excessive SMC activation. We recently reported that overexpression of the cAMP Response Element Binding Protein (CREB) transcription factor decreases SMC migration and proliferation. One of the genes downregulated by CREB expression likely to mediate a decrease SMC proliferation is the platelet-derived growth factor receptor-alpha (PDGFRa) gene. Animal studies demonstrate the PDFGRa expression is increased in the vessel wall in diabetes and insulin resistance (models where CREB content is decreased). The goals of the current proposal are to clarify molecular details of how changes in CREB and C/EBP delta in diabetes and insulin resistance affect gene expression using the PDGFRa as a model target gene. HYPOTHESIS: Loss of vascular CREB function and augmented C/EBPdelta function in diabetes promotes smooth muscle cell proliferation, in part, by increasing expression of PDGFRa. Manipulation of vascular CREB function will impact proliferative capacity and development of atherosclerosis. SPECIFIC AIMS: 1. To determine the impact of targeted vascular overexpression of active and inactive CREB on gene expression, mitogenic capacity and atherosclerosis, a. To examine SMC proliferation and PDFGRa expression in transgenic mice with vascular specific expression of CREB and dominant negative CREB (A-CREB). b To test the hypothesis that loss of CREB contributes to atherosclerotic burden using inducible expression of active and inactive CREB on an ApoE null background. 2. To employ the promoter of the PDGFRa gene as a model to define the regions/cis-acting elements in that are responsive to regulation through CREB and C/EBPdelta. a. Mutagenesis and Footprinting studies of the PDGFRa promoter, b. Confirm CREB and C/EBPdelta DNA binding using EMSA and Affinity purification, c. Identify in vivo occupancy using ChIP analysis of relevant CREB and C/EBP binding elements, d. Determine how PDGFRa expression is affected by changing concentrations and activation states of CREB and C/EBPdelta. 3. To determine whether PPARa mediated suppression of PDGFRa gene expression works through CREB and C/EBPs.

描述（由申请人提供）：我们实验室的长期目标是了解胰岛素抵抗和糖尿病期间发生的基因表达和调节的变化，这些变化允许过度SMC激活。我们最近报道，cAMP响应元件结合蛋白（CREB）转录因子的过表达降低了SMC迁移和增殖。 CREB表达下调的基因之一可能介导SMC增殖的降低是血小板衍生的生长因子受体-Alpha（PDGFRA）基因。动物研究表明，糖尿病和胰岛素抵抗的血管壁中PDFGRA表达增加（CREB含量降低的模型）。当前建议的目标是阐明糖尿病中CREB和C/C/EBP三角洲的变化以及胰岛素抵抗如何使用PDGFRA作为模型靶基因影响基因表达的分子细节。假设：糖尿病中血管CREB功能和增强C/EBPDELTA功能的丧失，部分通过增加PDGFRA的表达来促进平滑肌细胞的增殖。血管CREB功能的操纵将影响动脉粥样硬化的增殖能力和发展。具体目的：1。确定靶向血管过表达的活性和非活性Creb对基因表达，有丝分裂能力和动脉粥样硬化的影响，a。检查具有CREB和显性阴性CREB（A-CREB）的血管特异性表达的转基因小鼠中的SMC增殖和PDFGRA表达（A-CREB）。 B为了测试假设，即使用在APOE无效背景下的活动和非活性CREB的诱导表达诱导的表达有助于动脉粥样硬化负担。 2。将PDGFRA基因的启动子作为模型来定义通过CREB和C/C/EBPDELTA响应调节的区域/顺式作用元素。一个。 PDGFRA启动子的诱变和足迹研究，b。使用EMSA和亲和力纯化，c。确认CREB和C/EBPDELTA DNA结合。使用相关CREB和C/EBP结合元素的芯片分析识别体内占用率，d。确定PDGFRA表达如何受到CREB和C/C/EBPDELTA的浓度和激活状态的影响。 3。确定PPARA介导的PDGFRA基因表达是否通过CREB和C/EBP起作用。