Dual-Specificity Phosphatase 4 Overexpression in Cells Prevents Hypoxia/Reoxygenation-Induced Apoptosis via the Upregulation of eNOS.
Dual-Specificity Phosphatase 4 Overexpression in Cells Prevents Hypoxia/Reoxygenation-Induced Apoptosis via the Upregulation of eNOS.
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DOI：
10.3389/fcvm.2017.00022
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发表时间：
2017
影响因子：
3.6
通讯作者：
Chen CA
中科院分区：
文献类型：
--
作者：
Dougherty JA;Kilbane Myers J;Khan M;Angelos MG;Chen CA
丝裂原活化蛋白激酶（MAPK）信号级联调节几种细胞功能，包括分化、增殖、存活和凋亡。这些MAPK磷酸化的持续时间和幅度是其生理功能的决定性决定因素。双特异性磷酸酶对这些信号级联发挥动力学控制。以前，我们证明了DUSP 4 −/−心脏在离体心脏缺血/再灌注时，梗死面积更大，功能恢复较差。不受控制的p38激活和Nox 4表达的上调是这种功能改变的主要效应子。在这里，双特异性磷酸酶4（DUSP 4）在内皮细胞中的过表达被用来研究DUSP 4对活性氧（ROS）的产生和血管功能的调节作用，当细胞受到缺氧/复氧（H/R）损伤。用切割的caspase-3免疫染色显示，DUSP 4过表达阻止H/R后caspase-3活化和凋亡。通过调节p38活性、增加NO生物利用度和降低氧化应激发生有益作用。更重要的是，在H/R诱导的应激期间，DUSP 4过表达上调eNOS蛋白表达（1.62 ± 0.33对0.65 ± 0.16）。NO是参与调节血管张力、血管生长、血小板聚集和炎症调节的关键小分子。使用DUSP 4 -2荧光测定的NO产生水平表明，DUSP 4过表达增加NO产生，从而改善血管功能。使用二氢乙锭-HPLC法测定在经受H/R后从细胞产生的超氧化物的水平。结果表明，DUSP 4在细胞中的过表达减少了H/R诱导的超氧化物生成（1.56 ± 0.14对1.19 ± 0.05），从而减少了氧化应激。这也与总蛋白S-谷胱甘肽化的减少相关，S-谷胱甘肽化是蛋白氧化的指标。这些结果进一步支持了我们的假设，即DUSP 4是一种抗氧化基因，是氧化应激过程中调节MAPK，特别是p38的关键磷酸酶，其调节ROS的产生和eNOS的表达，从而保护免受氧化剂诱导的损伤或凋亡。总体而言，DUSP 4可能是治疗缺血性心脏病的一个很好的分子靶点。
Mitogen-activated protein kinases (MAPKs) signaling cascades regulate several cellular functions, including differentiation, proliferation, survival, and apoptosis. The duration and magnitude of phosphorylation of these MAPKs are decisive determinants of their physiological functions. Dual-specificity phosphatases exert kinetic control over these signaling cascades. Previously, we demonstrated that DUSP4−/− hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to ischemia/reperfusion. Uncontrolled p38 activation and upregulation of Nox4 expression are the main effectors for this functional alteration. Here, dual-specificity phosphatase 4 (DUSP4) overexpression in endothelial cells was used to investigate the role of DUSP4 on the modulation of reactive oxygen species (ROS) generation and vascular function, when cells were subjected to hypoxia/reoxygenation (H/R) insult. Immunostaining with cleaved caspase-3 revealed that DUSP4 overexpression prevents caspase-3 activation and apoptosis after H/R. The beneficial effects occur via modulating p38 activity, increased NO bioavailability, and reduced oxidative stress. More importantly, DUSP4 overexpression upregulates eNOS protein expression (1.62 ± 0.33 versus 0.65 ± 0.16) during H/R-induced stress. NO is a critical small molecule involved in regulating vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF-2 fluorescence demonstrated that DUSP4 overexpression augments NO production and thus improves vascular function. The level of superoxide generated from cells after being subjected to H/R was determined using dihydroethidium-HPLC method. The results suggested that DUSP4 overexpression in cells decreases H/R-induced superoxide generation (1.56 ± 0.14 versus 1.19 ± 0.05) and thus reduces oxidant stress. This also correlates with the reduction in the total protein S-glutathionylation, an indicator of protein oxidation. These results further support our hypothesis that DUSP4 is an antioxidant gene and a key phosphatase in modulating MAPKs, especially p38, during oxidative stress, which regulates ROS generation and eNOS expression and thus protects against oxidant-induced injury or apoptosis. Overall, DUSP4 may serve as an excellent molecular target for the treatment of ischemic heart disease.