Non-NF-kappa B elements are required for full induction of the rat type IInitric oxide synthase in vascular smooth muscle cells

1 We have investigated the role of the NF-kappa B binding sites and other p romoter elements beyond NF-kappa B in iNOS induction in rat vascular smooth muscle cells (SMC). 2 Rat aortic SMC transfected with iNOS promoter constructs with either muta tion or deletion of the downstream NF-kappa B site exhibited about 50% redu ction in promoter activity in response to a cytokine mixture, whereas eithe r mutation or deletion of the upstream NF-kappa B site reduced promoter act ivity by 90%, suggesting that the latter site is the most important, and th at co-existence of two NF-kappa B sites is necessary for iNOS induction. 3 Nuclear NF-kappa B activity was robustly induced by TNF-alpha. However, T NF-alpha alone did not induce iNOS promoter activity, protein expression, o r nitrite production, indicating that NF-kappa B activation alone is not su fficient for iNOS induction. 4 The construct up to -890 bp, containing the downstream NF-kappa B site, e xhibited little response to cytokines. The construct up to -1.0 kb, contain ing the two NF-kappa B sites exhibited only 22% of full promoter activity. The regions -1001 to -1368 bp and -2 to -2.5 kb contributed an additional 4 3 and 22% promoter activity, respectively. 5 Internal deletion or reversal of the orientation of -1001 to -1368 bp in the full promoter resulted in 40% reduction in promoter activity. 6 These data suggest that the co-existence of two NF-kappa B sites is essen tial for core promoter activity, but that full induction of the rat SMC iNO S gene requires other elements located between -1.0 to -1.37 and -2.0 to -2 .5 kb of the promoter.