title: Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis.
abstract: TNFα 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 TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα 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 TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-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 TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-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 TNFα receptor signaling to mitochondrial damage and apoptotic death.
authors.affiliation: Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195.
authors.forename: Calivarathan
authors.initials: C
authors.lastname: Latchoumycandane
authors.affiliation: Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195.
authors.forename: Gopal K
authors.initials: GK
authors.lastname: Marathe
authors.affiliation: Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195.
authors.forename: Renliang
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195. Electronic address: mcintyt@ccf.org.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: McIntyre
mesh_terms: D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000096:biosynthesis; D017209:Apoptosis / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D015227:Lipid Peroxidation / Q000502:physiology*; D008054:Lipid Peroxides / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D010743:Phospholipids / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D008054:Lipid Peroxides; D010743:Phospholipids; D014409:Tumor Necrosis Factor-alpha; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; C526455:Pafah2 protein, mouse