The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase. Activation by nuclear factor-kappa B.
The production of inducible nitric oxide synthase (iNOS) within vascular smooth muscle (VSM) cells following exposure to proinflammatory cytokines is a major cause of the vasorelaxation and hypotension of septic shock. We have defined the cytokine-responsive element of the murine iNOS promoter, transfected into a VSM cell line, and the role of the NF-kappa B/Rel family of proteins in iNOS gene activation in these cells. The combination of interleukin-1, interferon-gamma, and tumor necrosis factor-alpha stimulates promoter activity by a factor of 8.1-fold; single cytokines show little activity, while pairs of cytokines produce an intermediate effect. Using a series of promoter deletion mutants, we have defined the cytokine-responsive element from position -890 to -1002; this region contains an NF-kappa B-binding site as well as a number of interferon response elements. Nuclear proteins from cytokine-stimulated VSM cells which bind to an oligonucleotide containing this kappa B site are composed of p65 together with an unidentified protein of 50 kDa, which is not a known Rel family member. A promoter mutant with a 2-base pair change within this kappa B site, which abolishes NF-kappa B binding, has an activity of only approximately 34% (S.E. +/- 1.5) of the wild-type promoter. In addition, protein binding to this site is abolished by a specific inhibitor of NF-kappa B activation, which also abrogates iNOS activity. Residual inducibility in such mutant promoters is attributable to the presence of an independently functioning downstream kappa B site (-85 to -75). The mechanism by which NF-kappa B activates the iNOS promoter in VSM cells in response to cytokines appears to be markedly different to that operative in macrophages in response to lipopolysaccharide.
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D016328:NF-kappa B / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D051381:Rats; D013859:Thiocarbamates / Q000494:pharmacology; D014162:Transfection
D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D013859:Thiocarbamates; C066229:prolinedithiocarbamate; D011392:Proline; D019001:Nitric Oxide Synthase