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.
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