INVOLVEMENT OF 2 SP1 ELEMENTS IN BASAL ENDOTHELIAL PROSTAGLANDIN H-SYNTHASE-1 PROMOTER ACTIVITY

Prostaglandin H synthase-1 (PGHS-1) is a constitutively expressed key enzyme in the biosynthesis of physiologically important prostanoids. T he promoter of the human PGHS-1 gene lacks a TATA box, has a very GC-r ich region, and contains multiple transcription start sites, To identi fy the elements involved in the constitutive expression of the PGHS-1 gene, we constructed a 2075-base pair fragment (-2095 to -21 relative to the translation start codon) and a series of 5'-deletion mutants in to a promoterless luciferase expression vector, which was transfected in HUVEC. Two important regions were identified. DNase I footprinting identified a protected segment, which contains an Sp1 binding site pro ximal to the transcription start sites. Band shift assays confirmed sp ecific binding of Sp1 to this segment, Band shift assays further revea led specific binding of Sp1 to a distal region containing a canonical Sp1 site, Mutation of either Sp1 binding site significantly reduced th e promoter activity. When both sites were mutated, the activity was re duced to 29% of that of the wild type, Mutation of Sp1 sites did not a brogate promoter activity stimulated by phorbol ester. These results i ndicate that binding of Sp1 or its related proteins to two widely sepa rated Sp1 sites on the promoter region activates the basal PGHS-1 gene transcription.