REGULATION OF PDGF PRODUCTION BY THE ENDOTHELIUM

这个项目的中心假设是生长因子和 血管内皮细胞(EC)对病理生理刺激的趋化作用 凝血系统蛋白酶-α-凝血酶，在血管损伤和动脉粥样硬化过程中导致炎症反应。我们的具体目标是识别和表征凝血酶诱导内皮细胞血小板衍生生长因子(PDGF)表达增加的调节途径，并进一步建立依赖于内皮细胞PDGF表达的体内事件。这个项目是对上一个资助期进行的研究的直接延伸，在上一个资助期，我们发现了一种新的转录激活模式，这种模式是由潜在转录因子Y-box结合蛋白DBpB的mRNA释放凝血酶触发的，导致其裂解形式的运输 DBpB-205(205个氨基酸)结合到细胞核中。核DBpB-205结合凝血酶反应元件，调节靶基因的表达。此外，利用基因芯片的方法，我们发现凝血酶诱导了双特异性磷酸酶CL100/MKP-1，这可能在凝血酶调控的EC基因表达中发挥关键作用。我们计划在下一个供资期间继续通过实现三个目标来实现我们的总体目标。目的1是明确凝血酶诱导的内皮细胞中DBpB激活和靶基因表达的分子步骤，包括对DBpB-205的结构和功能的研究。目的2确定核双特异性磷酸酶CL100/MKP-1在凝血酶和溶血磷脂酸诱导PDGF B链形成中的作用。我们计划使用底物捕获技术来确定CL100/MKP-1的新底物。在目标3中，我们将确定EC特异性消融PDGF表达的体内后果。我们将检验这一假设，即仅通过EC阻止PDGF的表达就足以导致胚胎死亡，从而支持或驳斥EC来源的PDGF在胚胎发生中的重要性。我们计划在EC特异性Tie-1启动子的控制下，产生表达显性-负性PDGF A链突变形式的转基因小鼠。如果转基因小鼠成年，我们将确定这种EC特异性的PDGF消融对动脉粥样硬化斑块发展的影响。这些目的应该有助于阐明凝血酶和其他激动剂激活EC的潜在机制。

The central hypothesis of this project is that the inappropriate expression of growth factors and chemoattractants by endothelial cells (EC) in response to such pathophysiological stimuli as the coagulation system protease alpha-thrombin, leads to inflammatory responses during vascular injury and atherosclerosis. Our specific objective is to identify and characterize regulatory pathways induced by thrombin that lead to increased expression of platelet-derived growth factor (PDGF) by the endothelium and further to establish the in vivo events which are dependent on EC expression of PDGF. This project represents a direct extension of studies performed during the previous funding period in which we have identified a novel mode of transcriptional activation that is triggered by thrombin - release from mRNA of a latent transcription factor, the Y-box binding protein dbpB, leading to transport of its cleaved form dbpB-205 (205 amino acids) to the nucleus. Nuclear dbpB-205 binds the thrombin-response element and regulates target gene expression. Furthermore, using a gene array approach we have discovered that thrombin induces the dual-specificity phosphatase CL100/MKP-1, which may play a critical role in thrombin-regulated gene expression in EC. We plan to continue to approach our overall objective during the next funding period by pursuing three aims. Aim 1 is to define the molecular steps involved in thrombin-induced dbpB activation and target gene expression in EC, including structure-function studies on dbpB-205. Aim 2 is to define the role of the nuclear dual-specificity phosphatase CL100/MKP-1 in the induction of PDGF B chain by thrombin and by lysophosphatidic acid (lysoPA). We plan to identify novel substrates for CL100/MKP-1 using a substrate-capture technique. In Aim 3, we will identify the in vivo consequences of EC-specific ablation of PDGF expression. We will test the hypothesis that preventing PDGF expression by EC alone is sufficient to cause embryonic lethality, thus supporting or refuting the importance of EC-derived PDGF in embryogenesis. We plan to generate transgenic mice that are expressing a dominant-negative mutant form of PDGF A chain under the control of the EC-specific Tie-1 promoter. If the transgenic mice reach adulthood, we will determine the consequences of this EC-specific ablation of PDGF on atherosclerotic plaque development. These aims should help elucidate the mechanisms underlying EC activation by thrombin and by other agonists.