Molecular Regulation of the Bovine Endothelial Cell Nitric Oxide Synthase by Transforming Growth Factor–β 1

Abstract

The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor–β ₁ (TGFβ ₁ ). TGFβ ₁ plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGFβ ₁ might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGFβ ₁ increased ecNOS mRNA in a dose-dependent manner. TGFβ ₁ 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 TGFβ ₁ -treated cells. Transcriptional activity of the 5′-flanking promoter region of the ecNOS gene was increased by TGFβ ₁ , as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGFβ ₁ -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 in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGFβ ₁ . In conclusion, TGFβ ₁ 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.