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 o f ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor alpha (TNF alpha). In L9 29 cells, the addition of TNF alpha resulted in the release of arachid onate, which was followed by a prolonged accumulation of ceramide occu rring over 5-12 h and reaching 250% over base line. The formation of c eramide was accompanied by the hydrolysis of sphingomyelin and the act ivation of three distinct sphingomyelinases (neutral Mg2+-dependent, n eutral Mg2+-independent, and acidic enzymes). The variant cell line C1 2, which lacks cPLA(2), is resistant to the cytotoxic action of TNF al pha. 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 ca used 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 parti al restoration of TNF alpha-induced arachidonate release, ceramide acc umulation, and cytotoxicity. This study suggests that cPLA(2) is a nec essary component in the pathways leading to ceramide accumulation and cell death.