Estrogens increase transcription of the human endothelial NO synthase gene -: Analysis of the transcription factors involved

Estrogens have been found to reduce the incidence of cardiovascular disease that has been ascribed in part to an increased expression and/or activity of the vasoprotective endothelial NO synthase (NOS III). Some reports have shown that the level of expression of this constitutive enzyme can be upregulated by estrogens. The current study investigates the molecular mechanism of the NOS III upregulation in human endothelial EA.hy 926 cells. Incubation of EA.hy 926 cells with 17 beta-estradiol or the more stable 17 alpha-estradiol enhanced NOS III mRNA and protein expression up to 1.8-fold, without changing the stability of the NOS III mRNA. There was no enhancement of NOS III mRNA after incubation of EA.hy 926 cells with testoterone, progesterone, or dihydrocortisol or when 17 alpha-ethinyl estradiol was added together with the estrogen antagonist RU58668, indicating a specific estrogenic response. Nuclear run-on assays indicated that the increase in NOS III mRNA is the result of an estrogen-induced enhancement of NOS III gene transcription. In transient transfection experiments using a 1.6 kb human NOS III promoter fragment (which contains no bona fide estrogen-responsive element, ERE), basal promotor activity was enhanced 1.7-fold by 17 alpha-ethinyl estradiol. In electrophoretic mobility shift assay, nuclear extracts from estrogen-incubated EA.hy 926 cells showed no enhanced binding activity either for the ERE-like motif in the human NOS III promoter or for transcription factor GATA. However, binding of transcription factor Sp1 (which is essential for the activity of the human NOS III promoter) was significantly enhanced by estrogens. These data suggest that the estrogen stimulation of the NOS III promotor could be mediated in part an increased activity of transcription factor Sp1.