Direct sensing of endothelial oxidants by vascular endothelial growth factor receptor-2 and c-Src

• Published in: PloS one 2011-12, Vol.6 (12), p.e28454-e28454
• Main Authors: Lee, Monica, Choy, Wing C, Abid, Md Ruhul
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; Activation; AKT protein; Amino acids; Angiogenesis; Antibodies; Biology; Blotting, Western; Cells, Cultured; Cellular signal transduction; Coronary artery; Coronary Vessels - cytology; Coronary Vessels - metabolism; Cysteine; Endothelial cells; Endothelium; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Extracellular signal-regulated kinase; Fluorescent Antibody Technique; Genes, src - physiology; Heart surgery; Homeostasis; Hospitals; Humans; Immunofluorescence; Immunoprecipitation; Kinases; Localization; Medicine; NAD(P)H oxidase; NADPH Oxidases - antagonists & inhibitors; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; Nitric oxide; Oxidants; Oxidase; Oxidases; Oxidation; Oxidation-Reduction; Oxidizing agents; Oxygen; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Phosphotransferases; Proteins; Proto-Oncogene Proteins c-akt - metabolism; Rac1 protein; Reactive oxygen species; Reactive Oxygen Species - metabolism; Redox properties; RNA, Small Interfering - genetics; Signal Transduction; Signaling; siRNA; Src protein; Thiols; Transduction; Tyrosine; Vascular endothelial growth factor; Vascular Endothelial Growth Factor Receptor-2 - metabolism; Vascular endothelial growth factor receptors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0028454

ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis 'senses' NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs. Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47(phox) subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS. These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can 'sense' redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs.