on November 3, 2005
Published online before print November 3, 2005, doi: 10.1161/01.ATV.0000194074.59584.42
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Submitted on July 12, 2005
Accepted on October 20, 2005
Endothelial Nitric Oxide Synthase Activation by Tumor Necrosis Factor 
Through Neutral Sphingomyelinase 2, Sphingosine Kinase 1, and Sphingosine 1 Phosphate Receptors. A Novel Pathway Relevant to the Pathophysiology of Endothelium
Clara De Palma ;
From the Department of Pharmaco-Biology (C.D.P., C.P.), University of Calabria, Rende; Stem Cell Research Institute (C.D.P., C.P., E.C.), DIBIT-H San Raffaele Institute, Milan; the Department of Biochemical Sciences (E.M., P.B.), University of Firenze; E. Medea Scientific Institute (E.C.), Bosisio Parini; and the Department of Preclinical Sciences (E.C.), University of Milan, Italy.
* To whom correspondence should be addressed. E-mail: clementi.emilio{at}hsr.it.
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 S1P1 and S1P3, 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-.
Conclusions--This study establishes a previously undescribed link among TNF-, Sph1P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-.
Key words: endothelial NO synthase • TNF-
•
neutral sphingomyelinase 2 •
sphingosine kinase 1 •
sphingosine 1 phosphate
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