Dougherty Julie A, Kilbane Myers Joanna, Khan Mahmood, Angelos Mark G, Chen Chun-An
Department of Emergency Medicine, College of Medicine, The Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA.
Front Cardiovasc Med. 2017 Apr 24;4:22. doi: 10.3389/fcvm.2017.00022. eCollection 2017.
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 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.
丝裂原活化蛋白激酶（MAPKs）信号级联反应调节多种细胞功能，包括分化、增殖、存活和凋亡。这些MAPKs的磷酸化持续时间和程度是其生理功能的决定性因素。双特异性磷酸酶对这些信号级联反应进行动力学控制。此前，我们证明，当离体心脏遭受缺血/再灌注时，DUSP4基因敲除的心脏梗死面积更大，功能恢复较差。不受控制的p38激活和Nox4表达上调是这种功能改变的主要效应器。在此，当细胞遭受缺氧/复氧（H/R）损伤时，利用内皮细胞中双特异性磷酸酶4（DUSP4）的过表达来研究DUSP4对活性氧（ROS）生成调节和血管功能的作用。用裂解的caspase-3进行免疫染色显示，DUSP4过表达可防止H/R后caspase-3激活和细胞凋亡。这些有益作用通过调节p38活性、增加NO生物利用度和降低氧化应激而产生。更重要的是，在H/R诱导的应激过程中，DUSP4过表达上调eNOS蛋白表达（1.62±0.33对0.65±0.16）。NO是一种关键的小分子，参与调节血管张力、血管生长、血小板聚集和炎症调节。使用DAF-2荧光测定的NO生成水平表明，DUSP4过表达增加NO产生，从而改善血管功能。使用二氢乙锭-HPLC法测定细胞在遭受H/R后产生的超氧化物水平。结果表明，细胞中DUSP4过表达降低H/R诱导的超氧化物生成（1.56±0.14对1.19±0.05），从而降低氧化应激。这也与蛋白质氧化指标——总蛋白S-谷胱甘肽化的降低相关。这些结果进一步支持了我们的假设，即DUSP4是一种抗氧化基因，是氧化应激期间调节MAPKs（尤其是p38）的关键磷酸酶，其调节ROS生成和eNOS表达，从而防止氧化诱导的损伤或细胞凋亡。总体而言，DUSP4可能是治疗缺血性心脏病的一个极佳分子靶点。
