Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation
Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation
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DOI：
10.1371/journal.pone.0229948
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发表时间：
2020-03-10
期刊：
影响因子：
3.7
通讯作者：
Oyadomari, Seiichi
中科院分区：
文献类型：
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作者：
Hamada, Yoshimasa;Furumoto, Yuji;Oyadomari, Seiichi
整合应激反应（ISR）是最重要的细胞保护机制之一，由真核翻译起始因子2（eIF2α）的α亚基磷酸化所整合。四种eIF2α激酶，即血红素调节抑制因子（HRI）、双链RNA依赖的蛋白激酶（PKR）、PKR样内质网激酶（PERK）和通用控制非抑制蛋白2（GCN2），在多种应激条件下被激活。我们先前报道纳秒脉冲电场（nsPEFs）是一种激活ISR的潜在治疗工具。在本研究中，我们检测了哪种eIF2α激酶可被nsPEF处理所激活。为了评估起作用的eIF2α激酶，我们使用了先前建立的eIF2α激酶四重敲除（4KO）以及单个eIF2α激酶拯救的4KO小鼠胚胎成纤维细胞（MEF）。持续时间为70纳秒、电场强度为30 kV/cm的nsPEFs可导致野生型（WT）MEF细胞中eIF2α磷酸化。另一方面，nsPEF诱导的eIF2α磷酸化在4KO MEF细胞中完全消失，而通过HRI过表达得以恢复。CM - H2DCFDA染色显示nsPEFs产生活性氧物质（ROS），其激活了HRI。nsPEF诱导的eIF2α磷酸化可被活性氧清除剂N - 乙酰 - L - 半胱氨酸（NAC）处理所阻断。我们的结果表明，eIF2α激酶HRI负责nsPEF诱导的ISR激活，并且由nsPEF产生的ROS所激活。
The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the a subunit of eukaryotic translation initiation factor 2 (eIF2 alpha). Four eIF2 alpha kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2 alpha kinase is activated by nsPEF treatment. To assess the responsible eIF2 alpha kinase, we used previously established eIF2 alpha kinase quadruple knockout (4KO) and single eIF2 alpha kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2 alpha phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2 alpha phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2 alpha phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2a kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.