The central hypothesis of this project is that the inappropriate production of growth factors and chemoattractants by endothelial cells (EC) in response to pathophysiological stimuli, such as the coagulation system protease alpha-thrombin, leads to intimal hyperplasia of smooth muscle cells (SMC) during vascular injury and atherosclerosis. Our specific objective is to identify and characterize regulatory pathways induced by thrombin that lead to increased transcription of platelet- derived growth factor (PDGF) by the endothelium. This project represents a direct extension of studies performed during the previous funding period in which we have identified a thrombin-response element in the promoter of the PDGF B chain gene and have purified a potentially, novel trans-acting factor (thrombin-inducible clear factor or TNF) that binds to this DNA element. We are excited about these results since they represent both the identification of a novel intracellular pathway for PDGF induction distinct from that elicited by phorbol esters or injury, and the first mechanistic information about transcriptional regulation induced by thrombin in any cell type. We plan to continue to approach our overall objective during the next funding period by pursuing three aims.
In Aim 1, we propose to clone and characterize TINF, and perform structure/function studies with the goal of identifying this protein's DNA binding region and trans-activating domain. Stable transfectants of bovine EC expressing sense or antisense TINF will be prepared to examine the role of TINF in EC gene expression.
In Aim 2, we plan to elucidate the mechanism by which TINF is activated in EC in response to thrombin and compare the pathway of PDGF B chain induction by thrombin to the regulation of this gene by lysophosphatidylcholine (lysoPC) and the redox state of the EC. In the third aim, we propose to define the interaction of known and novel transcription factors that bind to the thrombin response element (ThRE). We will test whether sequences that are homologous to the ThRE in the promoters of other thrombin-responsive genes bind TINF and convey thrombin-responsiveness, and we plan to study the binding of TINF, Sp1 and Egr-1 to the ThRE. Finally, we propose to continue our use of the yeast one-hybrid system to identify other transcription factors that regulate the transcription of the PDGF B chain gene. Our pursuit of these aims should help elucidate the mechanisms underlying thrombin-induced EC activation. Our findings should also have direct relevance to the regulation of PDGF expression by other vascular cells, as well as by other diploid and neoplastic cells, and to the mechanism of action of thrombin in the many other cells that respond to this protease.
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