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

Abstract

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.

Nitric Oxide Differentially Regulates Induction of Type II Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells Versus Macrophages

“…NF-B activation is a necessary factor in iNOS induction. 27,32 NF-B complex I (Figure 3) was remarkably induced in RASMC and macrophages and consisted of heterodimers of the p65 and p50 subunits (Figure 3 and 4). However, consistently, NF-B complex II and III were mainly induced in RASMC, and these bands appear to contain p50 and other subunits of NF-B (Figure 3).…”

“…In macrophage cells, the downstream NF-B site at position Ϫ76 to Ϫ85 bp of the mouse iNOS promoter functions as a core promoter, 32,33 but the upstream NF-B site of the iNOS promoter plays an important role in eliciting responses to cytokines in the A7r5 rat smooth muscle cell line. 27 Similarly, when the iNOS promoter was transfected into cultured RASMC, a key region was located at Ϫ234 bp from the 5Ј-region. 28 Data have also demonstrated that LPS activation of the human iNOS promoter exhibits cell-type specificity.…”

In vivo regulation of inducible NO synthase in immune-mediated liver injury in mice

“…46,47 In vitro studies implicated that NF-B activation alone was fully responsible for induction of iNOS gene by lipopolysaccharide or cytokine in murine macrophages 41 and rat smooth muscle cell lines. 48 To evaluate the contribution of NF-B activation to con A-induced iNOS expression, PDTC, a highly selective inhibitor of NF-B, was used. 49 NF-B translocation was inhibited in PDTC-pretreated mice, indicating that NF-B is the main regulatory transcription factor in con A-mediated iNOS expression.…”

Suppression of Interleukin-1β-induced Nitric-oxide Synthase Promoter/Enhancer Activity by Transforming Growth Factor-β1 in Vascular Smooth Muscle Cells

“…Previous studies have demonstrated that the downstream NF-kBd site (Ϫ85 to Ϫ76) was necessary for iNOS inducibility by LPS in a macrophage-like cell line (RAW 264.7) (7-9). Recently, cytokine regulation of the iNOS promoter/enhancer has also been investigated (27) in a clonal cell line originating from the thoracic aorta of embryonic rats (A7r5) (28). Using multiple cytokine stimulation (IL-1␤, tumor necrosis factor-␣, and interferon-␥), these investigators (27) reported the upstream NF-B site (Ϫ971 to Ϫ962) may play a role in the regulation of the iNOS 5Ј-flanking sequence in A7r5 cells.…”