Abstract 17049: Dual-specificity Phosphatase-4 (DUSP4) Over-expression Prevents H/R-induced Apoptosis via the Up-regulation of eNOS
MAPK signaling cascades regulate several cellular functions, including differentiation, proliferation, and apoptosis. The duration and extent of phosphorylation of these MAPKs are critical determinants of their physiological effects. The kinetic control of these MAPK signal cascades is modulated by DUSPs. Previously, we demonstrated that DUSP4-/- hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to I/R. Uncontrolled p38 activation and upregulation of Nox4 expression are the main effectors for this functional alteration. In this study, DUSP4 overexpression in endothelial cells was used to investigate the role of DUSP4 in the modulation of ROS generation and vascular function, when cells were subjected to H/R insult. Immunostaining with cleaved caspse-3 revealed that DUSP4 overexpression prevents caspase-3 activation and apoptosis after H/R. The beneficial effects are via modulating p38 activity, eNOS upregulation, and increase in NO generation. More importantly DUSP4 overexpression upregulates eNOS expression and activation via S1177 phosphorylation 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 fluorescence microscopy demonstrated that DUSP4 overexpression augments NO generation, and thus improves vascular function. The level of superoxide generated from cells after being subjected to H/R was determined using DHE HPLC method. These results further support that DUSP4 overexpression in cells decreases H/R-induced superoxide generation (1.56 ± 0.14 versus 1.19 ± 0.05, *P < 0.05) and thus reduces oxidant stress. This also correlated to 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 provides protection against oxidant-induced injury or apoptosis. Therefore, DUSP4 is an excellent molecular target for the treatment of ischemic heart disease.
Author Disclosures: C. Chen: None. J. Kilbane Myers: None. M. Khan: None. M. Angelos: None.
- © 2016 by American Heart Association, Inc.