A reverse nuclear factor-kappaB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1beta and interferon-gamma in rat aortic smooth muscle cells.

摘要

The rat type II nitric oxide synthase (iNOS) promoter contains two nuclear factor-kappaB (NF-kappaB)-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-kappaB 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-kappaB site"). We hypothesized that the reverse NF-kappaB 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-1beta (IL-1beta, 100 U/ml) +/- interferon-gamma (IFN-gamma, 250 U/ml) for 30 min. IL-1beta, but not IFN-gamma, induced a DNA-protein complex that was supershifted by either anti-NF-kappaB p50 or anti-NF-kappaB p65 antibody. The functionality of the reverse NF-kappaB 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-kappaB 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-1beta-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-1beta- and IFN-gamma-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-kappaB heterodimer binds to a reverse-NF-kappaB site on the rat iNOS promoter and contributes to iNOS induction by IL-1beta and IFN-gamma in RASMCs.