Zhou Y, Li F, Tian X, Wang B, Ding M, Pang H
School of Forensic Medicine, China Medical University, Shenyang, People's Republic of China.
School of Forensic Medicine, China Medical University, Shenyang, People's Republic of China Department of Forensic Medicine, Baotou Medical College, Baotou, People's Republic of China.
Hum Exp Toxicol. 2014 Jul;33(7):761-71. doi: 10.1177/0960327113499044. Epub 2013 Oct 15.
The neurotoxin paraquat (PQ) causes apoptosis of dopaminergic neurons in mammalian cell culture and animal models, mimicking an important pathological feature of Parkinson's disease (PD). The phosphoinositide 3-kinase (PI3K)/Akt pathway is critical for several major survival signals in central nervous system neurons. Phosphatidylinositol 3-kinase 55 kDa gamma (p55PIK) is a regulatory subunit of PI3Ks with important roles in cell proliferation, antiapoptosis, and cell cycle progression. However, p55PIK involvement in mechanisms regarding progression and maintenance of neurodegenerative diseases is largely undetermined. We used PQ-induced apoptosis in human dopaminergic SH-SY5Y cells to investigate the association between p55PIK expression levels, subcellular location, and apoptosis. p55PIK expression was reduced in SH-SY5Y cells and p55PIK messenger RNA and protein expression levels were decreased after PQ treatment. Apoptosis induced by PQ was associated with caspase activation and decreased p55PIK expression. Restoration of p55PIK expression was observed after coincubation with a caspase inhibitor. Overexpressed full-length p55PIK in SH-SY5Y and human embryonic kidney 293 cells showed specific distribution in the nucleus and was cleaved in vitro by recombinant caspase 6 (C6), but not C3 and C7. A p55PIK construct lacking 24 N-terminal amino acids (N24) was tested for the presence of a potential C6-recognizable sequence and was found to express its proteins outside the nucleus. The results suggest that p55PIK may be involved in PQ-induced apoptosis signal transduction and that N24 is crucial for p55PIK subcellular localization. Thus, p55PIK could be a substrate of activated C6 during apoptosis, leading to loss of original biological functions and redistribution to disturb cell cycle progression.
神经毒素百草枯（PQ）在哺乳动物细胞培养和动物模型中可导致多巴胺能神经元凋亡，模拟帕金森病（PD）的一个重要病理特征。磷脂酰肌醇3激酶（PI3K）/Akt信号通路对中枢神经系统神经元的几个主要存活信号至关重要。磷脂酰肌醇3激酶55 kDaγ（p55PIK）是PI3Ks的一个调节亚基，在细胞增殖、抗凋亡和细胞周期进程中发挥重要作用。然而，p55PIK在神经退行性疾病进展和维持机制中的作用在很大程度上尚未明确。我们利用PQ诱导人多巴胺能SH-SY5Y细胞凋亡，以研究p55PIK表达水平、亚细胞定位与凋亡之间的关系。PQ处理后，SH-SY5Y细胞中p55PIK表达降低，p55PIK信使核糖核酸和蛋白质表达水平均下降。PQ诱导的凋亡与半胱天冬酶激活和p55PIK表达降低有关。与半胱天冬酶抑制剂共孵育后，观察到p55PIK表达恢复。在SH-SY5Y和人胚肾293细胞中过表达的全长p55PIK在细胞核中呈特异性分布，并在体外被重组半胱天冬酶6（C6）切割，但不被C3和C7切割。对缺失24个N端氨基酸（N24）的p55PIK构建体进行了潜在C6识别序列检测，发现其蛋白在细胞核外表达。结果表明，p55PIK可能参与PQ诱导的凋亡信号转导，且N24对p55PIK亚细胞定位至关重要。因此，p55PIK可能是凋亡过程中活化C6的底物，导致其丧失原有的生物学功能并重新分布，从而干扰细胞周期进程。
