Endothelial nitric oxide synthase activation by tumor necrosis factor alpha through neutral sphingomyelinase 2, sphingosine kinase 1, and sphingosine 1 phosphate receptors: a novel pathway relevant to the pathophysiology of endothelium.
OBJECTIVE
Tumor necrosis factor alpha (TNF-alpha), 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.


RESULTS
In human endothelial cells, we found that eNOS activation by TNF-alpha 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-alpha. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P1 and S1P3, either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-alpha 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-alpha.


CONCLUSIONS
This study establishes a previously undescribed link among TNF-alpha, Sph1P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-alpha.
De Palma
Clara
C
Department of Pharmaco-Biology, University of Calabria, Rende, Italy.
Meacci
Elisabetta
E
Perrotta
Cristiana
C
Bruni
Paola
P
Clementi
Emilio
E
D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000503:physiopathology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology; D000099157:Sphingosine Kinase
D002118:Calcium; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D051057:Proto-Oncogene Proteins c-akt; D049349:Receptors, Lysosphingolipid; D013108:Sphingomyelin Phosphodiesterase; D014409:Tumor Necrosis Factor-alpha; D000099157:Sphingosine Kinase; C496314:NOS3 protein, human; C491484:SMPD3 protein, human