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-alpha (TNF alpha) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNF alpha-treated L929 cells, its role in the TNF alpha-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A(2) (PLA(2)). To find out the mechanisms underlying the TNF alpha-induced release of AA, we investigated the relationship between TNF alpha stimulation and PLA(2) regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNF alpha and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking alpha type of cytosolic PLA(2) (cPLA(2)alpha)). Stimulation with TNF alpha/zVAD also caused the release of AA from L929-cPLA(2)alpha-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA(2)alpha) completely inhibited the TNF alpha-induced release of AA, but only partially inhibited the TNF alpha/zVAD -induced response in L929 cells. The TNF alpha/zVAD-induced release of AA from C12 and L929-cPLA(2)alpha-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA(2) activity, decreased the amount of AA released by TNF alpha/zVAD. TNF alpha/zVAD appears to stimulate release of AA from C12 cells in a cPLA(2)alpha-independent, BHA-sensitive manner. The possible roles of secretory PLA(2) and reactive oxygen species from different pools in the release of AA and cell death were discussed. (c) 2007 Elsevier Inc. All rights reserved.