Inhibition of Endothelial Vascular Cell Adhesion Molecule-1 Expression by Nitric Oxide Involves the Induction and Nuclear Translocation of IκBα

Abstract

The adhesion of circulating leukocytes to the vessel wall is an initiating event in atherogenesis and vascular inflammation (1, 2). Under certain conditions, the "activated" endothelium expresses cell surface adhesion molecules which mediate specific interactions between the endothelium and circulating leukocytes (3, 4). Factors that affect the induction of endothelial cell adhesion molecules such as vascular cell adhesion molecule (VCAM-1) 1 therefore may be important in regulating vascular inflammatory processes. Recent studies suggest that the activation of the pleotropic transcription factor nuclear factor-B (NF-B) is required for the transcriptional induction of endothelial cell adhesion molecules (5).The activation of NF-B involves the degradation of its cytoplasmic inhibitor, IB (6, 7). Presently, five distinct IB proteins have been shown to functionally retain NF-B in the cytoplasm and render it inactive (6). These IB proteins contain ankyrin repeat motifs that mask the nuclear localization sequence of NF-B subunits such as RelA (p65), c-Rel, and RelB (8, 9). Of the different IB proteins, the best described is IB␣. Following cytokine stimulation, IB␣ is phosphorylated by a novel ubiquitinated serine kinase (10). Phosphorylation of IB␣ targets the IB␣ for ubiquitination and rapid degradation by 26 S proteasomes (10, 11). The degradation of IB␣ then allows the unbound NF-B to translocate into the nucleus, where it can transactivate the enhancer elements of many proinflammatory genes (5).Modulation of IB␣ function and expression has been shown to regulate NF-B activation. For example, the phosphorylation of IB␣ is a key regulatory step in the activation of NF-B (11, 12). Indeed, recent studies indicate that salicylates and antioxidants inhibit NF-B and endothelial cell activation by preventing IB␣ phosphorylation and subsequent degradation (13-16). Alternatively, the nuclear accumulation of IB␣ resulting from overexpression of IB␣ or following stimulation with tumor necrosis factor (TNF)-␣ has been shown to displace NF-B from its cognate DNA and terminate NF-B-mediated transcriptional activity (17,18). Finally, some of the anti-inflammatory effects of glucocorticoids may be mediated through their inhibitory effects on NF-B, since glucocorticoids are known to induce IB␣ expression (19,20).We and others have shown that nitric oxide can inhibit NF-B and endothelial cell activation through non-cGMP-dependent mechanisms (21,22). Although NO donors appear to "stabilize" the NF-B-IB␣ heterotrimeric complex (23), the possibility that newly synthesized IB␣ could account for this stabilization has not been excluded. Furthermore, it is not known whether the induction of IB␣ expression by NO donors could actually lead to an increase in cytoplasmic or nuclear IB␣ protein levels. The purpose of this study, therefore, is to determine the mechanism(s) by which NO inhibits of NF-B activation and VCAM-1 expression in terms of its effects on IB␣ phosphorylation, expression, and nuclear accumulation.

NO Inhibits Cytokine-Induced iNOS Expression and NF-κB Activation by Interfering With Phosphorylation and Degradation of IκB-α

“…17 The same group has subsequently reported that GSNO did not prevent phosphorylation and degradation of I B-␣ 15 minutes after stimulation with TNF-␣ in the same cells, suggesting that NO inhibits NF-B activation by the late (2 hours) induction and nuclear translocation of I B-␣. 28 In the present study, however, NOR3 itself had no effect on basal I B-␣ expression during a 75-minute incubation period. The exact reasons for the apparent discrepancy between their reports and ours are unknown.…”

“…26,27 However, our results appear to be in contrast to 2 recent (though contradictory) reports from the same laboratory. 17,28 Liao and his associates have shown that NO donors inhibited TNF-␣-induced NF-B activation by induction and stabilization of I B-␣ in human endothelial cells; S-nitrosoglutathione (GSNO) prevented I B-␣ degradation 30 minutes after stimulation with TNF-␣. 17 The same group has subsequently reported that GSNO did not prevent phosphorylation and degradation of I B-␣ 15 minutes after stimulation with TNF-␣ in the same cells, suggesting that NO inhibits NF-B activation by the late (2 hours) induction and nuclear translocation of I B-␣.…”

Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity

“…We showed that induction of NOS2 by LPS suppressed the secretion of pulmonary Th2 cytokines while others have shown that inhibition of NO exacerbates airway hyperresponsiveness, eosinophilia, and C-C chemokines (21). 2) NO may inhibit the expression of cell adhesion molecules (VCAM-1) on endothelial cells that are mediated, in part, by inhibition of kB cis-acting elements) (45), which in turn might block the influx of inflammatory T cells and eosinophils to the lung. 3) NO may trigger intrapulmonary cell death.…”

Low‐energy laser irradiation increases endothelial cell proliferation, migration, and eNOS gene expression possibly via PI3K signal pathway

“…b-actin levels were used as a loading control (*P < 0.05, n ¼ 3). suppressing the inflammatory response induced by cytokines [48], inhibiting apoptosis [49], regulating cell migration, and angiogenesis [50,51]. Thus a stimulated increase in NO production by the EC in vitro could be a potential benefit, if such cells were used in lining a vascular graft.…”