Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-alpha (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, sphing osine kinase (SphK), in the sphingomyelin metabolic pathway resulting in pr oduction of sphingosine-1-phosphate (S1P) and that S1P is a potent antagoni st of TNF-mediated apoptosis, The TNF-induced SphK activation is independen t of sphingomyelinase and ceramidase activities, suggesting that TNF affect s this enzyme directly other than through a mass effect on sphingomyelin de gradation. In contrast to normal HUVEC, in a spontaneously transformed endo thelial cell line (C11) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addi tion of exogenous S1P or increasing endogenous S1P by phorbol ester markedl y protected C11 cell line from TNF-induced apoptosis, Conversely, N,N-dimet hylsphingosine, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF, Thus, me demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.