Functional Analysis of the Human Endothelial Nitric Oxide Synthase Promoter

To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS) gene expression, we have cloned the eNOS promoter and fused it to a luciferase reporter gene to map regions of the promoter important for basal transcription in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1 luciferase (LUC) (−1600 to +22 nucleotides) yielded a 35-fold increase in promoter. Progressive deletion from −1600 to −1033 (F2 and F3 LUC) did not significantly influence eNOS promoter activity. Further deletion from −1033 to −779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter activity, and still further deletion from −779 to −494 (F5 LUC) did not markedly influence activity. Deletion from −494 to −166 (F6 LUC) reduced eNOS promoter activity by 40-50%. Specific mutation of the consensus GATA site(−230) in the F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of GATA-2 to the oligonucleotide sequence containing the −230 GATA site. Next, we mutated the Sp1 site(−103) in the F3 and F6 LUC constructs and in the F3 GATA mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90% reduction in normalized luciferase activity. Gel shift and antibody supershift analysis using a BAEC nuclear extracts demonstrated four specific, DNA-protein complexes binding to the eNOS Sp-1 site, with the slowest migrating form composed of Sp1 and another nuclear protein. These data demonstrate that the Sp1 site is an important cis-element in the core eNOS promoter.

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