The Cytokine Responsive Vascular Smooth Muscle Cell Enhancer of Inducible Nitric Oxide Synthase

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.