Endothelial Nitric Oxide Synthase Activation by Tumor Necrosis Factor α Through Neutral Sphingomyelinase 2, Sphingosine Kinase 1, and Sphingosine 1 Phosphate Receptors

Abstract

Objective-Tumor necrosis factor ␣ (TNF-␣), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown. Methods and Results-In human endothelial cells, we found that eNOS activation by TNF-␣ is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and sphingosine-kinase-1 (SK1) and generation of sphingosine-1-phosphate (Sph1P). Inhibition of N-SMase2 inhibited Sph1P formation, whereas inhibition of SK1 did not affect N-SMase2 activation by TNF-␣. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P 1 and S1P 3 , either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-␣ via S1P receptors, activated by Sph1P generated through N-SMase2 and SK1 activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-␣. Key Words: endothelial NO synthase Ⅲ TNF-␣ Ⅲ neutral sphingomyelinase 2 Ⅲ sphingosine kinase 1 Ⅲ sphingosine 1 phosphate N itric oxide (NO) generated in the endothelium by the endothelial NO synthase (eNOS) plays crucial roles not only in physiology but also in regulating specific inflammatory events involving the endothelium, such as the expression of adhesion molecules and leukocyte adhesion. 1,2 Indeed, eNOS Ϫ/Ϫ animals, when exposed to inflammatory stimuli, display significant increases in extravasated neutrophils and poor wound healing and are more prone to diseases in which inflammation is significant, including atherosclerosis and diabetes. 1,2 eNOS regulates the effects on migration, angiogenesis, vasodilation, endothelial barrier permeability, apoptosis, and expression of inflammatory and adhesion molecules exerted by tumor necrosis factor ␣ (TNF-␣), a key proinflammatory cytokine acting on the endothelium. [3][4][5][6][7][8][9] In some cases, the effects of NO appeared directly associated with eNOS activity; 8,9 in others, they were consistent with the involvement of this enzyme, because they were too rapid to be explained by synthesis of the inducible NO synthase (NOS) 1,2 or because they took place in human cells 4,7 that do not express inducible NOS when exposed to TNF-␣ alone. 8,10 Despite the relevance to TNF-␣ action, eNOS activation by the cytokine in endothelial cells had been investigated in only 2 studies 8,11 that did not provide evidence of the pathway involved, apart from the identification of a phosphatidylinositol 3-kinase (PI3K)/Aktdependent step, 11 a signaling event common to many eNOSactivating stimuli. 2 In a recent study in HeLa cells, we reported that eNOS activation by TNF-␣ is mediated by the activation of neutral sphingomyelinase (N-SMase), with generation of the sphingolipid ceramid...

Tumor necrosis factor‐α exerts pro‐myogenic action in C2C12 myoblasts via sphingosine kinase/S1P2 signaling

“…So far, TNFa-stimulated SphK activity has been shown to be critical for its antiapoptotic [16] and mitogenic [17] action, as well as for the inflammatory reaction [9,10]. The present study demonstrates that TNFa signaling via SphK1 is implicated also in the regulation of skeletal muscle differentiation.…”

“…In view of the recognized ability of TNFa to regulate SphK [8][9][10] and the pro-myogenic role exerted by S1P and SphK in myoblasts [6,7], the effect of TNFa at low dose on SphK was then examined. Data illustrated in Fig.…”

Endothelial NOS is required for SDF-1α/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells

“…32 The amount of endothelial injury in the target organ and time course of local NO production after eNOS activation by Akt may play an important role for therapeutic stem cell treatment. 33 In human endothelial cells, the induction of eNOS by TNF-a has recently been described by De Palma et al 34 In contrast, a downregulation of eNOS was observed in association with high levels of TNF-a in patients with type 2 diabetes and microangiopathy. 35 Systemic L-NAME treatment and thereby …”

Sphingosine Kinase–Dependent Activation of Endothelial Nitric Oxide Synthase by Angiotensin II

“…In this report, the authors showed that silencing S1P 1 and/or S1P 3 receptors by means of siRNA prevents eNOS activation by tumor necrosis factor-␣. 40 To investigate whether S1P 1 and S1P 3 receptors are involved in the Ang II-induced NO production, we tested whether the novel S1P 1 /S1P 3 receptor antagonist VPC 23019 also augments the contractile effects of Ang II in the rat carotid artery, as seen for DMS and L-NNA. Indeed, VPC 23019, one of the few available S1P receptor antagonists, induced a significant increase in E max and a small, although not significant, leftward shift of the concentration-response curve for Ang II.…”