A Novel c-Jun-N-Terminal Kinase Pathway Stimulates High Output eNOS-derived NO in Inflamed Endothelium

High levels of nitric oxide (NO) generated in the vasculature under inflammatory conditions are usually attributed to inducible nitric oxide synthase (iNOS), but the role of the constitutively expressed endothelial NOS (eNOS) is unclear. In normal human lung microvascular endothelial cells (HLMVEC), bradykinin (BK) activates kinin B2 receptor (B2R) signaling that results in Ca2+-dependent activation of eNOS and transient NO. In inflamed HLMVEC (pretreated with interleukin-1β and interferon-γ), we found enhanced binding of eNOS to calcium-calmodulin at basal Ca2+ levels, thereby increasing its basal activity that was dependent on extracellular L-Arg. Furthermore, B2R stimulation generated prolonged high output eNOS-derived NO that is independent of increased intracellular Ca2+ and is mediated by a novel Gαi, MEK1/2 and JNK1/2-dependent pathway. This high output NO stimulated with BK was blocked with B2R antagonist, eNOS siRNA or eNOS inhibitor but not iNOS inhibitor. Moreover, B2R-mediated NO production and JNK phosphorylation were inhibited with MEK1/2 and JNK inhibitors or MEK1/2 and JNK1/2 siRNA but not with ERK1/2 inhibitor. BK induced Ca2+-dependent eNOS phosphorylation at Ser1177, Thr495 and Ser114 in cytokine-treated HLMVEC but these modifications were not dependent on JNK1/2 activation and were not responsible for prolonged NO output. These data suggest that JNK controls eNOS activation by a different mechanism, possibly via phosphorylation at a novel regulatory site. Mass spectrometry analysis revealed that cytokine treatment induced phosphorylation at Ser625 and Tyr735, while B2R stimulation promoted phosphorylation at Ser836, a predicted JNK phosphorylation site. We also found that B2R-mediated eNOS-derived NO production has opposing effects on wound healing and endothelial cell migration where in healthy endothelial cells BK is stimulatory but inhibitory under cytokine-treated conditions. Additionally, the reduction in endothelial cell migration was partially reversed in cells pre-incubated with a superoxide scavenger, tempol, thereby suggesting that the reduction in migration is mediated in part by oxidative and nitroxidative stress. Thus, eNOS-derived high output NO may impair angiogenesis and wound healing in inflammation. Understanding how JNK regulates prolonged eNOS-derived NO may provide new therapeutic approaches to ameliorate endothelial dysfunction caused by the overproduction of NO in inflammatory vascular or lung disease.