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 S-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.