The overall goal of this research proposal is to elucidate mechanisms by which the synthesis and the cellular levels of key prostanoid synthetic enzymes is regulated. We propose to concentrate on prostaglandin H synthase (PGHS), thromboxane A synthase (TXAS) and prostaglandin I synthase (PGIS) which catalyze the biosynthesis of TXA2 and PGI2. Based on our preliminary studies, we postulate that (1) Expression of PGHS-1 is governed by two promoter activities in the 5'- flanking region of its gene. Constitutive expression of PGHS-1 is driven by proximal promoter which is switched to a more powerful distal promoter when cells are stimulated by cytokines and phorbol esters, (2) Aspirin suppresses the distal promoter activity of PGHS-1 gene. and (3) TXAS expression is modulated at the posttranscriptional level by a truncated form of mRNA. To test these hypotheses, we propose three specific aims.
Specific Aim 1 is to characterize the promoter activities of the PGHS-1 and PGHS-2 genes and to evaluate how they are regulated by IL-1 and phorbol esters. By expressing chimeric plasmids containing varying length of 5' flanking region of PGHS-1 conjugated to a luciferase cDNA as reporter gene, the proximal and distal promoter elements will be defined by using 5'- deletion mutants coupled with site-directed mutagenesis. The mechanism by which these two promoters ar utilized to drive PGHS-1 transcription will be elucidated. We will determine whether a discrete repressor is present and whether it suppresses the distal promoter activity. We will characterize the PGHS-2 gene by similar approaches. We will evaluate whether salicylates cause suppression of distal and/or proximal promoters and elucidate the mechanism by which they exert such an action. We will also test whether salicylates influence the promoter activity of the PGHS-2 gene.
Specific Aim 2 is to purify PGIS protein from bovine aorta, obtain N-and internal peptide sequences and clone PGIS cDNA from our HUVEC cDNA library. We will express the cDNA in baculovirus expression system and COS-1 cells and characterize the protein expressed in these cells. We will then evaluate the regulation of PGIS MRNA levels in endothelial cells and correlate it with PGIS protein level and enzyme activities.
Specific Aim 3 is to study the expression of two forms of TXAS mRNA in a megakaryoblastic cell line, MEG=01 cell during PMA-induced differentiation of this cell line. We will evaluate whether the truncated form of mRNA (TXAS-2) downregulates TXAS protein production by co-expressing these 2 forms of cDNa in COS-1 cells and/or baculovirus expression system. We will continue our work to delineate the structure of the TXAS gene and characterize its promoter activity. These investigations will provide novel and important information regarding the molecular mechanisms of TXA2 and PGI2 synthesis and will be valuable for studying their role s in vascular thrombosis and vascular diseases.
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