Phospholipase D protects ECV304 cells against TNFα-induced apoptosis

Tumor necrosis factor α (TNFα), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFα-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFα-induced apoptosis. TNFα/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1 h) whereas both PLD activity and PLD1 protein were strongly decreased after 24 h. TNFα/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFα-induced cell death.