Tumor necrosis factor-alpha upregulated PHLPP1 through activating nuclear factor-kappa B during myocardial ischemia/reperfusion
Tumor necrosis factor-alpha upregulated PHLPP1 through activating nuclear factor-kappa B during myocardial ischemia/reperfusion
复制标题
心肌缺血/再灌注期间肿瘤坏死因子-α 通过激活核因子-κ B 上调 PHLPP1
DOI：
10.1016/j.lfs.2018.06.023
复制
发表时间：
2018-08-15
期刊：
影响因子：
6.1
通讯作者：
Xing, Yuan
中科院分区：
文献类型：
--
作者：
Tang, Bin;Ma, Jing;Xing, Yuan
目的：富亮氨酸重复序列的普列克底物蛋白同源结构域蛋白磷酸酶1（PHLPP1）特异性调节蛋白激酶B（PKB，Akt）的磷酸化丝氨酸473位点，在心肌缺血/再灌注（I/R）过程中对抗细胞存活。先前的研究表明PHLPP1的表达水平受多种机制控制。然而，心肌I/R后心脏PHLPP1表达的调节机制仍不清楚。
主要方法：本研究利用体内心肌I/R损伤的小鼠模型以及体外新生大鼠心室肌细胞（NRVMs）缺氧/复氧（H/R）损伤模型。通过蛋白质印迹法测定PHLPP1、核因子 - κB（NF - κB）和磷酸化核因子 - κB（pNF - κB）的表达。通过免疫荧光法评估PHLPP1的表达以及NF - κB的转位。染色质免疫沉淀（ChIP）实验用于检测NF - κB与phlpp1基因启动子区域的结合。
关键发现：心肌I/R在I/R（30分钟/2小时）后对心脏PHLPP1表达无影响，但在再灌注4小时后降低。在体外，H/R（4小时/1小时）和肿瘤坏死因子 - α（TNF - α）刺激导致NRVMs中PHLPP1上调，这可被依那西普阻断。然而，过氧化氢（H₂O₂）诱导的氧化应激在早期对NRVMs的PHLPP1表达无明显影响，但N - 乙酰半胱氨酸（NAC）预处理在H₂O₂刺激4小时后增加了PHLPP1水平。TNF - α和H/R导致NF - κB的表达和转录活性增加，同时伴有PHLPP1的高表达。吡咯烷二硫代氨基甲酸酯（PDTC），一种NF - κB抑制剂，不仅在TNF - α处理的心肌细胞中，而且在H/R处理组中都阻止了这种反应。
意义：这些结果表明TNF - α参与复氧过程中心脏PHLPP1的上调，这是由NF - κB转录活性介导的。
Aims: The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) specifically regulates phospho-Ser473 of protein kinase B (PKB, Akt) opposing cell survival during myocardial ischemia/reperfusion (I/R). Previous studies demonstrated PHLPP1 expression level was controlled by several mechanisms. However, the regulation mechanism of cardiac PHLPP1 expression following myocardial I/R remains unknown.Main methods: The current study utilized the mouse model of myocardial I/R injury in vivo and the neonatal rat ventricular myocytes (NRVMs) of hypoxia/reoxygenation (H/R) injury in vitro. Expression of PHLPP1, nuclear factor-kappa B (NF-kappa B) and pNF-kappa B were determined by western blot. The expression of PHLPP1 and translocation of NF-kappa B was assessed by immunofluorescence. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NF-kappa B to the promoter region of phlpp1 gene.Key findings: Myocardial I/R had no effect on cardiac PHLPP1 expression following I/R (30 min/2 h) but decreased after 4 h reperfusion. In vitro, H/R (4 h/1 h) and tumor necrosis factor-alpha (TNF-alpha)-stimulation resulted in upregulation of PHLPP1 in NRVMs, which was blocked with etanercept. Yet, H2O2-induced oxidative stress had no obvious effect on PHLPP1 expression of NRVMs at early stage but N-acetylcysteine (NAC) pretreatment increased PHLPP1 levels after 4 h H2O2 stimulation. TNF-a and H/R led to both expression and transcriptional activity of NF-kappa B,accompany with higher expression of PHLPP1. Pyrrolidine dithiocarbamate (PDTC), a NF-kappa B inhibitor, prevented the response not only in TNF-alpha-treated cardiomyocytes but also in H/Rtreated group.Significance: These results implicated that TNF-alpha involved in cardiac PHLPP1 upregulation during reoxygenation, which was mediated by NF-kappa B transcriptional activity.