Release of arachidonic acid induced by tumor necrosis factor-α in the presence of caspase inhibition: Evidence for a cytosolic phospholipase A2α-independent pathway

Stimulation of L929 cells with tumor necrosis factor-α (TNFα) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFα-treated L929 cells, its role in the TNFα-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A2 (PLA2). To find out the mechanisms underlying the TNFα-induced release of AA, we investigated the relationship between TNFα stimulation and PLA2 regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFα and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking α type of cytosolic PLA2 (cPLA2α)). Stimulation with TNFα/zVAD also caused the release of AA from L929-cPLA2α-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA2α) completely inhibited the TNFα-induced release of AA, but only partially inhibited the TNFα/zVAD-induced response in L929 cells. The TNFα/zVAD-induced release of AA from C12 and L929-cPLA2α-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA2 activity, decreased the amount of AA released by TNFα/zVAD. TNFα/zVAD appears to stimulate release of AA from C12 cells in a cPLA2α-independent, BHA-sensitive manner. The possible roles of secretory PLA2 and reactive oxygen species from different pools in the release of AA and cell death were discussed.