The overall goal of this project is to understand at the molecular level how eicosanoid biosynthesis is regulated in health and disease. In the present proposal, we will focus on the regulatory mechanisms underlying the synthesis of key enzymes in the cyclooxygenase pathway that catalyzes the biosynthesis of thromboxane A and prostacyclin. From novel, interesting data derived from our preliminary studies, we postulate that (1) transcription of PGHS-1 gene is governed by two promoters. The constitutive expression is driven by the proximal promoter while IL-1 and PMA-induced stimulation of PGHS-1 transcription is driven by the more powerful distal promoter; (2) aspirin and other salicylates suppress the distal promoter activity of PGHS-1 gene and (3) two forms of thromboxane A synthase (TXAS) mRNA are expressed in monocytes. To test these hypotheses, we propose 4 specific aims.
Specific aim 1 is to evaluate the differential expression of PGHS-1 and PGHS-2 in cultured human endothelial cells. Temporal relation between the mRNA levels and protein levels and enzyme activities in response to IL-1 and phorbol ester stimulation will be determined. Potential roles of eicosanoids as feedback, regulators will be elucidated.
Aim 2 is to extent our earlier observations that aspirin and sodium salicylate suppress PGHS-1 mRNA and protein levels. We will determine the differential suppression of PGHS-1 and 2 gene expression by salicylates in vitro and in vivo. In both specific aims, the mRNA levels will be quantified by reverse transcription - PCR(RT-PCR) assays.
Specific aim 3 is to characterize the promoter activities of PGHS-1 and PGHS-2 genes and to evaluate how they are regulated by stimulants and pharmacological agents. We will use luciferase as a reporter gene to define the proximal and distal cis- acting elements utilizing 5-deletion fragments coupled with site-directed mutagenesis. We will elucidate the mechanism by which these 2 promoters are utilized to drive transcriptions and to determine whether a discrete repression is present to suppress the distal promoter activity. We will characterize PGHS-2 gene by similar approaches.
Specific aim 4 is to assess the expression of two forms of TXAS mRNA in blood monocytes and THP-1 cells during PMA-induced differentiation. We will evaluate whether expression of TXAS-2, a truncated form of TXAS-1 downregulates the synthesis of TXAS protein. These studies will provide new, valuable information regarding the regulation of prostanoid synthetic enzymes and the role of these enzymes in control of TXA2, and PGI2 synthesis in cerebrovascular thrombosis and stroke.
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