Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany; German Cancer Consortium (DKTK), Partner Site Frankfurt, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany.
Biochem Pharmacol. 2018 Jul;153:242-247. doi: 10.1016/j.bcp.2018.01.025. Epub 2018 Jan 11.
Mouse embryonic fibroblasts (MEFs) have extensively been used to study necroptosis, a recently identified form of programmed cell death. However, very little is yet known about the role of necroptosis and its regulation by reactive oxygen species (ROS) in cell types naturally exposed to high oxygen levels such as mouse lung fibroblasts (MLFs). Here, we discover that MLFs are highly susceptible to undergo necroptosis in a ROS-dependent manner upon exposure to a prototypic death receptor-mediated necroptotic stimulus, i.e. cotreatment with tumor necrosis factor (TNF)α, Smac mimetic and the caspase inhibitor zVAD.fmk (TSZ). Kinetic analysis revealed that TSZ rapidly induces cell death in MLFs. Pharmacological inhibition of receptor-interacting protein kinase (RIPK)1 by necrostatin-1 (Nec-1) or RIPK3 by GSK'872 significantly rescues TSZ-stimulated cell death. Also, genetic silencing of RIPK3 or mixed lineage kinase domain-like pseudokinase (MLKL) significantly protects MLFs from TSZ-mediated cell death. Prior to cell death, TSZ significantly increases production of ROS. Importantly, addition of radical scavengers such as butylated hydroxyanisole (BHA) or α-Tocopherol (α-Toc) significantly suppresses TSZ-induced cell death in parallel with a significant reduction of ROS generation. Consistently, BHA prevented TSZ-triggered phosphorylation of MLKL similar to the addition of GSK'872. Thus, our study demonstrates for the first time that MLFs are prone to undergo necroptosis in response to a prototypic necroptotic stimulus and identifies ROS as important mediators of TSZ-triggered necroptosis.
小鼠胚胎成纤维细胞（MEFs）已广泛用于研究细胞程序性死亡的一种新形式——坏死性凋亡。然而，对于自然暴露在高氧环境中的细胞类型（如小鼠肺成纤维细胞，MLFs）中坏死性凋亡及其活性氧物质（ROS）调节作用的了解却很少。在这里，我们发现 MLFs 在受到典型死亡受体介导的坏死性凋亡刺激物（即 TNFα、Smac 模拟物和半胱天冬酶抑制剂 zVAD.fmk 的联合处理）作用时，以 ROS 依赖的方式高度易发生坏死性凋亡。动力学分析显示，TSZ 可迅速诱导 MLFs 细胞死亡。用坏死性凋亡抑制剂-1（Nec-1）抑制受体相互作用蛋白激酶 1（RIPK1）或 GSK'872 抑制 RIPK3，可显著挽救 TSZ 刺激的细胞死亡。此外，RIPK3 或混合谱系激酶结构域样伪激酶（MLKL）的基因沉默也可显著保护 MLFs 免受 TSZ 介导的细胞死亡。在细胞死亡之前，TSZ 可显著增加 ROS 的产生。重要的是，加入自由基清除剂如丁基羟基茴香醚（BHA）或 α-生育酚（α-Toc）可显著抑制 TSZ 诱导的细胞死亡，同时显著减少 ROS 的产生。同样，BHA 可阻止 TSZ 触发的 MLKL 磷酸化，与 GSK'872 的添加效果相似。因此，本研究首次证明 MLFs 易对典型的坏死性凋亡刺激物发生坏死性凋亡，并确定 ROS 是 TSZ 触发的坏死性凋亡的重要介质。
