Suppression of interleukin-1beta-induced nitric-oxide synthase promoter/enhancer activity by transforming growth factor-beta1 in vascular smooth muscle cells. Evidence for mechanisms other than NF-kappaB.
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)-beta1 down-regulates inducible NOS after its induction by interleukin (IL)-1beta 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-1beta or vehicle. IL-1beta 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-1beta-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-1beta stimulation. Mutation of the NF-kappaB site within this region partially reduced IL-1beta-inducible activity; however, a large portion of activity remained independent of the NF-kappaB site. TGF-beta1 suppressed promoter/enhancer activity after IL-1beta stimulation, and this suppression was complete in the construct with a mutated NF-kappaB site. In addition, TGF-beta1 did not decrease the binding of nuclear proteins to the NF-kappaB site. These data suggest that the ability of TGF-beta1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-kappaB motif in vascular smooth muscle cells.
Perrella
M A
MA
Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Patterson
C
C
Tan
L
L
Yet
S F
SF
Hsieh
C M
CM
Yoshizumi
M
M
Lee
M E
ME
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000187:drug effects*; D007375:Interleukin-1 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D017931:DNA Primers; D007375:Interleukin-1; D016328:NF-kappa B; D009687:Nuclear Proteins; D016212:Transforming Growth Factor beta; D019001:Nitric Oxide Synthase