Zhou Hui-Ren, Lau Allan S, Pestka James J
Departments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-1224, USA.
Toxicol Sci. 2003 Aug;74(2):335-44. doi: 10.1093/toxsci/kfg148. Epub 2003 May 28.
Trichothecene mycotoxins and other protein synthesis inhibitors activate mitogen-activated protein kinase (MAPKs) via a mechanism that has been termed the "ribotoxic stress response." MAPKs are believed to mediate the leukocyte apoptosis that is observed following experimental exposure to these chemical agents in vitro and in vivo. The purpose of this research was to test the hypothesis that double-stranded, RNA-activated protein kinase R (PKR) is a critical upstream mediator of the ribotoxic stress response induced by the trichothecene deoxynivalenol (DON) and other translational inhibitors. DON was found to readily induce phosphorylation of JNK 1/2, ERK 1/2, and p38 in the murine macrophage RAW 264.7 cell line, within 5 min of culture addition, in a concentration-dependent fashion. Effects were maximal from 15 to 30 min and lasted up to 6 h. The translational inhibitors anisomycin and emetine also had similar effects when added to cultures at equipotent concentrations to DON. DON rapidly activated PKR within 1 to 5 min, as evidenced by autophosphorylation and by phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Interestingly, the latter effect was associated with rapid degradation of eIF2alpha. Pretreatment of RAW 264.7 cells with two inhibitors of PKR, 2-aminopurine (2-AP) or adenine (Ad), markedly impaired MAPK phosphorylation in RAW 264.7 cells according to the following rank order JNK>p38>ERK. The capacity of DON to induce MAPK phosphorylation was also markedly suppressed in a stable transformant of the human promonocytic U-937 cell line containing an antisense PKR expression vector. This suppression followed a rank order of JNK>p38>ERK in this PKR-deficient cell line when compared to control cells transfected with vector only. Apoptosis induction by DON and two other translational inhibitors, anisomycin and emetine, was almost completely abrogated in PKR-deficient cells. Together, the results indicate that PKR plays a critical upstream role in the ribotoxic stress response inducible by translational inhibitors.
单端孢霉烯族毒素和其他蛋白质合成抑制剂通过一种被称为“核糖体毒性应激反应”的机制激活丝裂原活化蛋白激酶（MAPK）。MAPK被认为介导了在体外和体内实验性暴露于这些化学物质后观察到的白细胞凋亡。本研究的目的是检验以下假设：双链RNA激活蛋白激酶R（PKR）是由单端孢霉烯族脱氧雪腐镰刀菌烯醇（DON）和其他翻译抑制剂诱导的核糖体毒性应激反应的关键上游介质。发现在培养物添加后5分钟内，DON能以浓度依赖的方式在小鼠巨噬细胞RAW 264.7细胞系中迅速诱导JNK 1/2、ERK 1/2和p38的磷酸化。作用在15至30分钟时达到最大，并持续长达6小时。当以与DON等效的浓度添加到培养物中时，翻译抑制剂茴香霉素和吐根碱也有类似的作用。DON在1至5分钟内迅速激活PKR，这通过自身磷酸化以及真核起始因子2α（eIF2α）的磷酸化得以证明。有趣的是，后一种作用与eIF2α的快速降解有关。用两种PKR抑制剂2-氨基嘌呤（2-AP）或腺嘌呤（Ad）预处理RAW 264.7细胞，根据以下顺序JNK>p38>ERK，显著损害了RAW 264.7细胞中的MAPK磷酸化。在含有反义PKR表达载体的人原单核细胞U-937细胞系的稳定转化体中，DON诱导MAPK磷酸化的能力也被显著抑制。与仅用载体转染的对照细胞相比，在这个PKR缺陷细胞系中，这种抑制遵循JNK>p38>ERK的顺序。在PKR缺陷细胞中，DON和其他两种翻译抑制剂茴香霉素和吐根碱诱导的凋亡几乎完全被消除。总之，结果表明PKR在翻译抑制剂诱导的核糖体毒性应激反应中起关键的上游作用。
