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

Calivarathan Latchoumycandane, Gopal K. Marathe, Renliang Zhang, Thomas M. McIntyre
2012 Journal of Biological Chemistry  
Reactive oxygen species (ROS) produced by TNF␣ induce apoptosis, but how this occurs and the actual molecules that damage mitochondria are undefined. Results: Molecular manipulation of phospholipid peroxidation and oxidatively truncated phospholipid degradation shows that oxidized phospholipids are essential for TNF␣-induced cell death. Conclusion: Oxidatively truncated phospholipids couple membrane cytokine stimulation to mitochondrial apoptosis. Significance: Fragmented phospholipids are
more » ... enous ROS products that cause cell death. TNF␣ 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␣ stimulation in sufficient quantities to induce apoptosis and if they are essential in TNF␣-induced cytotoxicity. We found that TNF␣ 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␣ exposure, whereas ROS inhibition abolished Az-PC accumulation and TNF␣-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNF␣-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␣-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNF␣induced apoptosis because overexpression of PAFAH2 (a phospholipase A 2 that selectively hydrolyzes truncated phospholipids) blocked TNF␣-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential ele-ment connecting TNF␣ receptor signaling to mitochondrial damage and apoptotic death. Tumor necrosis factor ␣ (TNF␣) is a master regulator of inflammatory and immune signaling (1) , where its complex signals vary over time (2), with prolonged stimulation promoting extrinsic apoptosis through caspase activation (3). This induces cleavage of the BH3 domain-only member of the Bcl2 family Bid that promotes intrinsic mitochondria-dependent apoptosis (4) through release of cytochrome c to form an activated apoptosome (5). TNF␣-induced apoptosis involves oxidizing radicals as intermediaries (6, 7), but neither the precise way TNF␣ couples cytokine stimulation (either at the plasma membrane or after internalization (8)) to cell death nor the precise role of reactive oxygen species (ROS) 3 in cell death is known. ROS are involved in a myriad of inflammatory, immunologic, and cell signaling events. Participation of ROS in the cytotoxicity of TNF␣ is firmly established (7, 9, 10) , but the transient nature of radicals and their interconversion, along with a general lack of specific tools to identify specific compounds, combine to obfuscate understanding of how ROS actually cause cell death in TNF␣-exposed cells. TNF␣ stimulates ROS production from NADPH oxidase (9) that, depending on cell type, employs the non-phagocytic NOx1 (6) or NOx4 (11) NADPH oxidase complex. Polyunsaturated fatty acyl residues of membrane phospholipids are energetically favored ROS targets (12), producing phospholipid (hydro)peroxides. These peroxidized phospholipids are also products of the 12/15-lipoxygenase of several cells (13, 14) . Chemical (13) reduction or enzymatic (15) reduction of these unstable oxidatively modified phospholipids by glutathione peroxidase-4 (GPx4) protects against the toxic effects of oxidative stress (16, 17), so these lipids are components of TNF␣induced toxicity. What remains unexplained, however, is the
doi:10.1074/jbc.m111.300012 pmid:22433871 pmcid:PMC3366783 fatcat:qi5nkbgui5a3lkxf5quo23lozm