Activation of Endothelial Nitric-Oxide Synthase by Tumor Necrosis Factor-α: A Novel Pathway Involving Sequential Activation of Neutral Sphingomyelinase, Phosphatidylinositol-3′ kinase, and Akt

Abstract

Activation of endothelial nitric-oxide synthase (eNOS) has been shown to occur through various pathways involving increases in the cytosolic Ca 2ϩ concentration, activation of the phosphatidylinositol-3Ј kinase/Akt pathway, as well as regulation by other kinases and by protein-protein interactions. We have recently reported that eNOS, expressed in an inducible HeLa Tet-off cell line, is activated by tumor necrosis factor-␣ (TNF-␣) in a previously undescribed pathway that involves the lipid messenger ceramide. We have now characterized this pathway. We report here that eNOS activation in response to TNF-␣ correlated with phosphorylation of Akt at Ser 473 and of eNOS itself at Ser 1179. Akt and eNOS phosphorylation, as well as eNOS activation, were blocked by inhibitors of both phosphatidylinositol-3Ј kinase and neutral sphingomyelinase. In contrast, although acid sphingomyelinase was also stimulated by TNF-␣, its inhibition was without effect. The activation of neutral sphingomyelinase triggered by TNF-␣ was insensitive to phosphatidylinositol-3Ј kinase inhibitors. Taken together, these results indicate that eNOS activation by TNF-␣ occurs through sequential activation of neutral sphingomyelinase and of the phosphatidylinositol-3Ј kinase/Akt pathway. The time course of eNOS activation induced through this pathway was markedly different from that triggered by ATP and epidermal growth factor, which activate eNOS through an increase in intracellular Ca 2ϩ concentration and through a sphingomyelinase-independent stimulation of the phosphatidylinositol-3Ј kinase/Akt pathway, respectively. The novel pathway of activation of eNOS described here may have broad biological relevance because neutral sphingomyelinase is activated not only by TNF-␣ but also by a variety of other physiological and pathological stimuli.

Altered nitric oxide/cGMP platelet signaling pathway in platelets from patients with acute coronary syndromes

“…One of the triggers of this phenomeon may be the proinflammatory cytokine TNF-α, as its circulating levels were significantly increased in both UA and AMI patients [8]. Indeed, TNF-α is known to promote platelet aggregation in patients with heart failure [20], and has been recently shown to stimulate eNOS phosphorylation via the PI3K/Akt signal transduction pathway [13,1,7].…”

“…Given these latest findings, the current concept of NO signalling needs to be revised highlighting the plausible biphasic role of NO in platelet activation; at the low concentrations, produced by platelet eNOS, NO would promote platelet secretion and aggregation, while at higher concentrations NO would inhibit platelet activation [15,17]. 1 …”

Recent advances in the immunobiology of ceramide

“…NOS III activation can involve ceramide generation by either acid or neutral sphinomyelinases, stimulated either by basic fibroblast growth factor (bFGF) in CHO-K1 cells (Goldkorn et al, 1998). It can also involve tumor necrosis factor-α (TNF-α) in HeLa cell clones transfected with NOS III under a tetracycline-responsive element (Barsacchi et al, 2003;Bulotta et al, 2001). NOS III activation by TNF-α requires stimulation of the phosphatidylinositol 3 kinase (PI3K)/Akt pathway while bFGF activated NOS III is independent of PI3/Akt activation.…”

Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3′,5′-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells

“…The conditions and concentrations of use of the various compounds interfering with the SMase/ceramide and NO signaling pathways have been described in detail previously (31,33,36,37,39). In brief, cell incubations with D609, manumycin A, scyphostatin, DETA-NO, 8 Br-cGMP, ODQ, KT5823, C2 ceramide, and exogenous A-SMase were performed for 10 min and those with imipramine for 1 h before LPS or E. coli administration.…”

“…To inhibit A-SMase, we used imipramine, which induces proteolysis of the enzyme (31,(33)(34)(35), and D609, which inhibits the phosphatidylcholine-specific phospholipase C, an enzyme known to be involved in A-SMase activation by LPS and other stimuli through generation of diacylglycerol (22,23,31,33,39,43). As controls of specificity we used scyphostatin and manumycin A, which are inhibitors of the neutral SMase (39,44,45). When administered alone, none of the compounds had any effect on basal sphingomyelin hydrolysis (not shown).…”