Suppression of Interleukin-1β-induced Nitric-oxide Synthase Promoter/Enhancer Activity by Transforming Growth Factor-β1 in Vascular Smooth Muscle Cells

Nitric-oxide synthases (NOS) utilize L-arginine to produce NO, a potent vasodilator that contributes to the regulation of vascular tone. We demonstrated previously that transforming growth factor (TGF)-β1 down-regulates inducible NOS after its induction by interleukin (IL)-1β by decreasing the rate of inducible NOS gene transcription. In the present study we transfected reporter plasmids containing various lengths of the inducible NOS 5′-flanking region into primary cultured rat aortic smooth muscle cells and stimulated the cells with IL-1β or vehicle. IL-1β increased the activity of the plasmid containing −1485 to +31 of the inducible NOS gene by more than 10-fold, indicating the presence of IL-1β-responsive elements. Further deletion analysis revealed that a construct containing −234 to +31 of the inducible NOS gene contained the majority of promoter/enhancer activity after IL-1β stimulation. Mutation of the NF-κB site within this region partially reduced IL-1β-inducible activity; however, a large portion of activity remained independent of the NF-κB site. TGF-β1 suppressed promoter/enhancer activity after IL-1β stimulation, and this suppression was complete in the construct with a mutated NF-κB site. In addition, TGF-β1 did not decrease the binding of nuclear proteins to the NF-κB site. These data suggest that the ability of TGF-β1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-κB motif in vascular smooth muscle cells.