Abstract 15314: Mechanisms of p22phox C242T Polymorphism in Reduced TNF Alpha-Induced Nox2 Activation in Endothelial Cells
The NADPH oxidase (Nox) by generating reactive oxygen species (ROS) is involved in the pathogenesis of many cardiovascular diseases. Several clinical studies report a single nucleotide polymorphism (SNP) of p22phox (C242T), a key component of the Nox enzyme, is negatively associated with the incidence of coronary heart disease. However, the mechanism involved remains unknown. In this study we combined computer molecular modelling with cell and molecular biology to investigate the potential mechanism of the p22phox C242T on endothelial ROS production. Our in silico 3D protein structure model of p22phox consists of an N-terminal transmembrane domain (124 a.a.) and a C-terminal cytoplasmic domain (71 a.a.). The p22phox C242T SNP causes a substitution of His72 to Tyr72 in its extracellular loop, resulting in significant morphological changes in the putative interactive region of the p22phox with catalytic subunit (Nox2), and this may result in reduced ROS production. To test this hypothesis, we generated a p22phox C242T construct by site-directed mutagenesis and transfected human pulmonary microvascular endothelial cells (HPMEC) with wild-type or C242T vectors. Compared to wild-type control, C242T expression had no significant effect on basal ROS production. However, when cells were challenged with TNFα (100U/ml, 45min), C242T expression significantly reduced (53±5.6%) levels of ROS production detected by both chemiluminescence and DHE fluorescence. Reduced ROS production was accompanied with inhibited TNFα-induced ERK1/2 and p38MAPK activation and NFκB phosphorylation. Furthermore, C242T expression reduced significantly (28.9±2%) the TNFα (24h)-induced endothelial cell apoptosis detected by Annexin-V flow-cytometry and significantly inhibited monocyte adhesion (52.42±4.3%). In conclusion, we reported for the first time that p22phox C242T SNP is associated with a significant protein morphological change affecting Nox2 activity and results in reduced TNFα-stimulated ROS production in endothelial cells. Our study provides important insight into the mechanism of this SNP and its clinical relevance as a natural inhibitory factor of the incidence of coronary heart disease.
- © 2012 by American Heart Association, Inc.