Oxidatively Truncated Phospholipids Are Required Agents of Tumor Necrosis Factor α (TNFα)-induced Apoptosis

TNF alpha generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the ( hydro) peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNF alpha stimulation in sufficient quantities to induce apoptosis and if they are essential in TNF alpha-induced cytotoxicity. We found that TNF alpha induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNF alpha exposure, whereas ROS inhibition abolished Az-PC accumulation and TNF alpha-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNF alpha-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNF alpha-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNF alpha- induced apoptosis because overexpression of PAFAH2 ( a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNF alpha-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNF alpha receptor signaling to mitochondrial damage and apoptotic death.