Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

Monica Lee,Md Ruhul Abid,Wing C Choy,Soumitro Pal

doi:10.1371/journal.pone.0028454

Abstract

BackgroundADPH 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.Methodology/Principal FindingsUsing 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 p47phox 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.Conclusion/SignificanceThese 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.

Highlights

Reactive oxygen species (ROS) are oxidizing molecules that have unpaired electrons, such as superoxide (O2?-), hydroxyl anion (HO?), and nitric oxide (NO?), or that may not have free electrons but possess oxidizing ability, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite (ONOO-)
Generalized reduction in reactive oxygen species (ROS) levels in human coronary artery ECs (HCAEC) using diphenylamine (DPI, 10 mM, for 30 mins) resulted in selective inhibition of Vascular endothelial growth factor (VEGF)-mediated activation of c-Src and Akt but not PLCc-1 or ERK1/2. These findings suggest that ROS play a crucial role in selective propagation of signals from VEGFR-2 to downstream c-Src-phosphatidyl inositol 3-kinase (PI3K)-Akt but not PLCc-1ERK1/2
Using a biotinylated probe that binds proteins with cysteine sulfenic acid modification, we show for the first time that VEGF induces thiol oxidation in VEGFR-2 and c-Src in HCAEC

Summary

Introduction

Reactive oxygen species (ROS) are oxidizing molecules that have unpaired electrons, such as superoxide (O2?-), hydroxyl anion (HO?), and nitric oxide (NO?), or that may not have free electrons but possess oxidizing ability, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite (ONOO-). ROS, at physiological concentration, have recently been shown to be essential for signal transduction in vascular cells, including endothelial cells (ECs) [4,5,6,7,8,9,10,11,12,13]. NADPH oxidase-derived ROS have been implicated in EC proliferation, migration, and angiogenesis [14,15,17]. ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). 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

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