MOLECULAR REGULATION OF THE BOVINE ENDOTHELIAL-CELL NITRIC-OXIDE SYNTHASE BY TRANSFORMING GROWTH FACTOR-BETA(1)

The promoter region of the endothelial cell nitric oxide synthase (ecN OS) gene contains potential response elements for transforming growth factor-beta(1) (TGF beta(1)). TGF beta(1) plays an important role in t he pathogenesis of atherosclerosis, vascular hypertrophy, and angiogen esis. We therefore sought to determine whether TGF beta(1) might modul ate ecNOS expression in bovine aortic endothelial cells (BAEC). TGF be ta(1) increased ecNOS mRNA in a dose-dependent manner. TGF beta(1) als o increased ecNOS protein content. The production of nitrogen oxides ( NOx), assessed by chemiluminescence, and nitric oxide synthase activit y, assessed by arginine/citrulline conversion, were increased in TGF b eta(1)-treated cells. Transcriptional activity of the 5'-flanking prom oter region of the ecNOS gene was increased by TGF beta(1), as assesse d by transfection with promoter/luciferase constructs. Deletion analys is suggested that the TGF beta(1)-response element was present between nucleotides -1269 and -935 from the first transcription start site, i n which a putative nuclear factor-1 (NF-1) binding site existed. Gel s hift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding s ite in a sequence-specific manner. Mutation of the putative NF-1 bindi ng site in the promoter/luciferase construct significantly decreased t he responsiveness to TGF beta(1). In conclusion, TGF beta(1) increases ecNOS expression associated with an increase in production of NO in B AEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.