A reverse nuclear factor-κB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1β and interferon-γ in rat aortic smooth muscle cells

The rat type II nitric oxide synthase (iNOS) promoter contains two nuclear factor-κB (NF-κB)-binding sites, one upstream (-965 to -956 bp) and one downstream (-107 to -98 bp), which are important for iNOS induction. We have identified a third NF-κB site located at -901 to -892 bp whose sequence is identical to that of the upstream site but with the opposite orientation ('the reverse NF-κB site'). We hypothesized that the reverse NF-κB site, like the other two sites, is important for iNOS induction. With the use of a rat iNOS promoter fragment of -906 to -887 bp as probe, electrophoretic mobility shift assays were performed on nuclear proteins extracted from rat aortic smooth muscle cells (RASMCs) treated with interleukin-1β (IL-1β, 100 U/ml) ± interferon-γ (IFN-γ, 250 U/ml) for 30 min. IL-1β, but not IFN-γ, induced a DNA-protein complex that was supershifted by either anti-NF-κB p50 or anti-NF-κB p65 antibody. The functionality of the reverse NF-κB site was evaluated by mutation experiments and transfection assays. The wild-type and mutated -1.4 kb rat iNOS promoter-luciferase constructs were transfected into RASMCs. Compared with the wild type, reverse-NF-κB site (-901 to -892 bp) deletion, substitution of T for C at -894 bp, and substitution TTT for CCC at -896 to -894 bp decreased the IL-1β-induced promoter activity by 67% (p < 0.001), 45% (p < 0.001), and 56% (p < 0.001), respectively. These deletion/substitutions also decreased the IL-1β- and IFN-γ-induced promoter activity by 74% (p < 0.001), 53%(p < 0.001), and 63% (p < 0.001), respectively. In conclusion, a p50 and p65 NF-κB heterodimer binds to a reverse-NF-κB site on the rat iNOS promoter and contributes to iNOS induction by IL-1β and IFN-γ in RASMCs. (C) 2000 Elsevier Science Inc.