Factors that increase synthesis of the PGH synthase enzyme appear to do so by increasing transcription of the PGH synthase gene. We have isolated and mapped genomic clones of the mouse PGH synthase gene and have sequenced approximately 2200 bp on the 5'-side of the transcriptional start site. We have identified consensus enhancer sequences related to AP-1 (which mediate increased transcription by Jun/Fos in response to phorbol esters, serum and growth factors) and to the dioxin responsive element (DRE) (which mediates increased transcription by the Aromatic hydrocarbon (Ah) receptor in response to polycyclic aromatic hydrocarbons (dioxin/TCDD, PCB, PBB). We have also identified a putative negative glucocorticoid regulatory element (nGRE) that may downregulate transcription of the gene in response to glucocorticoids. We propose to test whether the AP-1, DRE and nGRE sequences regulate PGH synthase gene transcription and to determine whether other sequences, different from known enhancer elements, are present which regulate transcription of the PGH synthase gene.
Specific Aims #1 and #3 are to prepare plasmids containing 5'-flanking sequences of the PGH synthase gene adjoined to a chloramphenicol acetyltransferase (CAT) reporter gene and to use these plasmids in transfection assays to determine if the AP-1 and DRE consensus sequences or any other sequences in the 5'-flanking region of the gene enhance transcription of the PGH synthase gene in response to serum-, PDGF-, or phorbol ester-stimulation.
Specific Aim #2 is to use the PGHS-CAT vectors constructed for Specific aims #1 and #2 to determine if the negative-glucocorticoid responsive element that we have identified in the 5'-flanking sequence of the PGH synthase gene, or any other region of the flanking sequence, can mediate transcriptional inhibition by glucocorticoids via glucocorticoid-receptor dependent inhibition of basal, PDGF-, serum-, or TPA-stimulated transcription of the PGH synthase gene.
Specific Aim #4 is to determine if a Splice Variant of PGH synthase lacking amino acids 523-585 (including the active site serine) exhibits peroxidase activity. A pSVT7 expression vector containing the cDNA for this splice variant will be constructed, transfected into cos-1 cells, and tested for peroxidase activity.
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