Non-NF-kappaB elements are required for full induction of the rat type II nitric oxide synthase in vascular smooth muscle cells.
We have investigated the role of the NF-kappaB binding sites and other promoter elements beyond NF-kappaB in iNOS induction in rat vascular smooth muscle cells (SMC). Rat aortic SMC transfected with iNOS promoter constructs with either mutation or deletion of the downstream NF-kappaB site exhibited about 50% reduction in promoter activity in response to a cytokine mixture, whereas either mutation or deletion of the upstream NF-kappaB site reduced promoter activity by 90%, suggesting that the latter site is the most important, and that co-existence of two NF-kappaB sites is necessary for iNOS induction. Nuclear NF-kappaB activity was robustly induced by TNF-alpha. However, TNF-alpha alone did not induce iNOS promoter activity, protein expression, or nitrite production, indicating that NF-kappaB activation alone is not sufficient for iNOS induction. The construct up to -890 bp, containing the downstream NF-kappaB site, exhibited little response to cytokines. The construct up to -1.0 kb, containing the two NF-kappaB sites exhibited only 22% of full promoter activity. The regions -1001 to -1368 bp and -2 to -2.5 kb contributed an additional 43 and 22% promoter activity, respectively. Internal deletion or reversal of the orientation of -1001 to -1368 bp in the full promoter resulted in 40% reduction in promoter activity. These data suggest that the co-existence of two NF-kappaB sites is essential for core promoter activity, but that full induction of the rat SMC iNOS gene requires other elements located between -1.0 to -1.37 and -2.0 to -2.5 kb of the promoter.
Zhang
H
H
Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, GA 30912-2500, USA. hzhang@mail.mcg.edu
Teng
X
X
Snead
C
C
Catravas
J D
JD
D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000502:physiology; D004790:Enzyme Induction; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat