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

1. We have investigated the role of the NF-κB binding sites and other promoter elements beyond NF-κB in iNOS induction in rat vascular smooth muscle cells (SMC). 2. Rat aortic SMC transfected with iNOS promoter constructs with either mutation or deletion of the downstream NF-κB site exhibited about 50% reduction in promoter activity in response to a cytokine mixture, whereas either mutation or deletion of the upstream NF-κB site reduced promoter activity by 90%, suggesting that the latter site is the most important, and that co-existence of two NF-κB sites is necessary for iNOS induction. 3. Nuclear NF-κB activity was robustly induced by TNF-α. However, TNF-α alone did not induce iNOS promoter activity, protein expression, or nitrite production, indicating that NF-κB activation alone is not sufficient for iNOS induction. 4. The construct up to -890 bp, containing the downstream NF-κB site, exhibited little response to cytokines. The construct up to -1.0 kb, containing the two NF-κB 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. 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-κB 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.