Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-β₁

The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor- β₁ (TGFβ₁). TGFβ₁ plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGFβ₁ might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGFβ₁ increased ecNOS mRNA in a dose-dependent manner. TGFβ₁ also increased ecNOS protein content. The production of nitrogen oxides (NO(x)), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion, were increased in TGFβ₁-treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGFβ₁, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGFβ₁-response element was present between nucleotides -1269 and - 935 from the first transcription start site, in which a putative nuclear factor-1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGFβ₁. In conclusion, TGFβ₁ increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.