Abstract
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.
Highlights
Nitric-oxide synthases (NOS) utilize L-arginine to produce Nitric oxide (NO), a potent vasodilator that contributes to the regulation of vascular tone
We have previously demonstrated that interleukin (IL)-1 and tumor necrosis factor-␣, two important cytokines downstream of endotoxin in the cascade of events leading to septic shock [15, 16], induce isoform of NO synthase (iNOS) mRNA by increasing gene transcription in vascular smooth muscle cells [5]
The luciferase activity produced by iNOS(Ϫ1485/ϩ31) in the presence of IL-1 is comparable with that produced by pGL2-Control, which is driven by the potent SV40 enhancer and promoter
Summary
Cell Culture—RASMC were harvested from male Sprague-Dawley rats (200 –250 grams) by enzymatic dissociation according to the method of Gunther et al [18]. The construct containing bp Ϫ1485 to ϩ31 was inserted into pGL2Basic in the reverse orientation, and this construct was named iNOS(ϩ31/Ϫ1485 R) Another PCR-generated fragment of the iNOS 5Ј-flanking sequence, containing bp Ϫ234 to Ϫ53, was inserted into pGL2-Promoter to assess its effects on heterologous promoter activity in the presence of IL-1. The constructs containing the mutated NF-Bd site, iNOS(Ϫ1485/ϩ31 NF-Bm) and iNOS(Ϫ331/ϩ31 NFBm), were inserted into pGL2-Basic, and their orientation and sequences were confirmed by the dideoxy chain termination method [19]. Statistics—Comparisons between groups were made by factorial analysis of variance followed by Fisher’s least significant difference test when appropriate
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