Transcriptional Regulation of PDGF Receptor Alpha

描述(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控的变化，这些变化允许SMC过度激活。我们最近报道，cAMP反应元件结合蛋白(CREB)转录因子的过表达降低了SMC的迁移和增殖。CREB表达下调可能介导SMC增殖减少的基因之一是血小板衍生生长因子受体-α(PDGFRA)基因。动物研究表明，在糖尿病和胰岛素抵抗(CREB含量降低的模型)中，PDFGRa在血管壁上的表达增加。目前这项提案的目标是以PDGFRA作为模型靶基因，阐明糖尿病和胰岛素抵抗中CREB和C/EBP Delta的变化如何影响基因表达的分子细节。假设：糖尿病患者血管CREB功能丧失和C/EBPDelta功能增强，部分是通过增加PDGFRA的表达来促进平滑肌细胞的增殖。血管CREB功能的调控将影响动脉粥样硬化的增殖能力和发展。具体目的：1.研究血管特异表达CREB和显性阴性CREB(A-CREB)转基因小鼠的SMC增殖和PDFGRa表达，以确定CREB活性和非活性CREB在血管靶向过表达对基因表达、促有丝分裂能力和动脉粥样硬化的影响。B通过在载脂蛋白E缺失的背景上诱导活性和非活性CREB的表达来检验CREB缺失对动脉粥样硬化负担的影响的假设。2.以PDGFRA基因启动子为模型，确定通过CREB和C/EBPDelta调控的区域/顺式作用元件。A.PDGFRA启动子的突变和足迹研究，b.用EMSA和亲和纯化证实CREB和C/EBPDelta的DNA结合，c.通过芯片分析相关的CREB和C/EBP结合元件来鉴定体内占位，d.确定改变CREB和C/EBPDelta的浓度和激活状态如何影响PDGFRA的表达。3.确定PPARa是否通过CREB和C/EBPs抑制PDGFRA基因的表达。

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.