Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195.
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195.
J Biol Chem. 2012 May 18;287(21):17693-17705. doi: 10.1074/jbc.M111.300012. Epub 2012 Mar 20.
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.
TNFα 在细胞表面产生活性氧 (ROS)，诱导细胞死亡，但 ROS 如何与线粒体通讯以及它们的特定凋亡作用尚不清楚。ROS 氧化磷脂生成易损的氢过氧化物，并在（氢）过氧化物功能附近发生片段化，形成短链磷脂，如己二酰基磷脂酰胆碱 (Az-PC)。Az-PC 具有相对的可溶性，外源性 Az-PC 可迅速进入细胞，破坏线粒体完整性并启动内在凋亡。我们确定在 TNFα 刺激期间细胞内是否形成了足够数量的这种有毒磷脂以诱导凋亡，以及它们在 TNFα 诱导的细胞毒性中是否必不可少。我们发现 TNFα 诱导 Jurkat 细胞中 ROS 的形成和磷脂过氧化，化学抑制 NADPH 氧化酶活性或 siRNA 抑制 NADPH 氧化酶-4 亚基均可阻止 ROS 积累和磷脂过氧化。质谱分析表明，TNFα 暴露后，磷脂过氧化物和随后的 Az-PC 增加，而 ROS 抑制则消除了 Az-PC 的积累和 TNFα 诱导的细胞死亡。谷胱甘肽过氧化物酶-4 (GPx4)，可特异性代谢脂质过氧化物，在 TNFα 刺激的细胞死亡前下降。过表达 GPx4 克服了这一点，减少了过氧化磷脂的积累，阻止了 Az-PC 的积累，并防止了死亡。相反，GPx4 siRNA 敲低增强了磷脂过氧化，增加了 TNFα 刺激的 Az-PC 形成和凋亡。短链磷脂是 TNFα 诱导凋亡的必需因素，因为过表达 PAFAH2（一种选择性水解短链磷脂的磷脂酶 A2）可阻断 TNFα 诱导的 Az-PC 积累，而不影响磷脂过氧化。PAFAH2 也消除了凋亡。因此，磷脂氧化和断裂为凋亡性磷脂是连接 TNFα 受体信号与线粒体损伤和凋亡死亡的必需因素。
