Identification of arachidonic acid as a mediator of sphingomyelin hydrolysis in response to tumor necrosis factor α

A sphingomyelin (SM)-signaling cycle has been described in human leukemia- derived HL-60 cells (Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080). Activation of the cycle by tumor necrosis factor α (TNFα) occurs rapidly, with peak levels of approximately 30% SM hydrolysis observed within 45-60 min. The mechanisms by which TNFα induces this SM turnover remain largely unexplored. In this study, arachidonic acid (AA) was investigated as a potential mediator of TNFα effects on SM turnover. In HL-60 cells, 30 nM TNFα stimulated the release of AA within 5- 10 min. In turn, AA stimulated SM hydrolysis and concomitant ceramide generation within 20 min of addition to cells. Other fatty acids, notably oleate, mimicked the effects of AA on SM hydrolysis, but the methyl ester and alcohol analogs of fatty acids were inactive. Diacylglycerol, a candidate mediator of TNFα responses, failed to stimulate SM hydrolysis even at concentrations as high as 300 μM. Moreover, in an in vitro assay, AA activated a cytosolic sphingomyelinase dose dependently, with 10-100 μM AA inducing 3-4-fold activation, thus suggesting a direct effect of AA on sphingomyelinase. Melittin, a potent phospholipase A₂ activator, induced SM hydrolysis at concentrations as low as 35 nM. However, unlike AA, melittin was unable to stimulate sphingomyelinase activation in an in vitro assay system. Finally, exogenous addition of AA also produced antiproliferative effects reminiscent of ceramide effects. Thus, a role for the phospholipase A₂/AA pathway in mediating TNFα induction of the SM cycle is supported by multiple lines of evidence. These studies begin to elucidate a mechanism of TNFα signaling and identify a close relationship between glycerophospholipid and sphingolipid signaling. AA, therefore, may be pivotal to understanding the sphingomyelin-signaling cascade.