Phospholipase A(2) is necessary for tumor necrosis factor alpha-induced ceramide generation in L929 cells

The role of cytosolic phospholipase A(2) (cPLA(2)) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor alpha (TNF alpha). In L929 cells, the addition of TNF alpha resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5-12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg2+-dependent, neutral Mg2+-independent, and acidic enzymes). The variant cell line C12, which lacks cPLA(2), is resistant to the cytotoxic action of TNF alpha. TNF alpha was able to activate nuclear factor kappa B in both the wild-type L929 cells and the C12 cells. However, TNF alpha was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C-6-ceramide overcame the resistance to TNF alpha and caused cell death in C12 cells to a level similar to that in L929 cells, The introduction of the cPLA(2) gene into C12 cells resulted in partial restoration of TNF alpha-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA(2) is a necessary component in the pathways leading to ceramide accumulation and cell death.