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. 2011;6(12):e28454.
doi: 10.1371/journal.pone.0028454. Epub 2011 Dec 1.

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

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Free PMC article

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

Monica Lee et al. PLoS One. .
Free PMC article

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusion/significance: 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. VEGF-induced activation of c-Src, Akt, but not VEGFR-2, PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS.
(A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47phox were subject to Western blots as described in Materials and methods. Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.
Figure 2
Figure 2. c-Src and VEGFR-2 are oxidized in VEGF-treated HCAEC in the presence of NADPH oxidase-derived ROS.
(A) Schematic presentation depicting cysteinyl-labeling assay to determine oxidative modification in intracellular proteins. Non-oxidized protein thiols are alkyalated by IAA, oxidized thiols are reduced back to SH-moiety by DTT and subsequently biotinylated by IAP. Biotinylated proteins are then pulled-down by streptavidin-agarose followed by Western blots. (B) Upper panel: cysteinyl labeling assay to Identify thiol oxidation of proteins in VEGF-treated (50 ng/ml for 2 mins) HCAEC lysates using biotinylated IAP probe. HCAEC were transfected with Scram-si or si-p47phox as indicated. After cell lysis in the presence of IAA followed by DTT treatment and IAP labeling, 1.5 mg biotinylated protein lysates were subject to immunoprecipitation using Streptavidin-agarose beads and immunoblotted using anti-VEGFR-2 and anti-c-Src antibodies. Lower panel: Western blot for VEGFR-2 using 50 µg of parallel HCAEC lysates as loading control. (B) Quantitative analyses of oxidized VEGFR-2 (upper panel) and c-Src (lower panel). Bar graph shows quantitative densitometric analysis of three independent cysteinyl labeling assays (as in A) using NIH J image (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant.
Figure 3
Figure 3. VEGF-induced interaction between VEGFR-2 and c-Src requires NADPH oxidase-derived ROS.
(A) Co-immunoprecipitation (co-IP) assay using 1.2 mg protein lysates of HCAEC that were transfected with Scram-si or si-p47phox and treated without or with VEGF (50 ng/ml for 5 min). IP was carried out using anti-VEGFR-2 antibody followed by immunoblotting using anti-c-Src (upper panel) and anti-VEGFR-2 (lower panel) antibodies. (B) Quantitative analyses of VEGFR-2-bound c-Src. Bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant.
Figure 4
Figure 4. VEGF induces subcellular co-localization of c-Src and internalized VEGFR-2 in an ROS-dependent manner.
HCAEC transfected with control (Scram-si) (A) or si-p47phox (B) were immunofluorescently double labeled for internalized VEGFR-2 (green) and c-Src (red). VEGFR-2 on HCAEC was labeled with single chain E-tagged antibody (scFvA7, Fitzerald) as described in Materials and methods. After incubation with VEGF (50 ng/ml for 10 min), in order to remove the antibody from the cell surface, cells were placed on ice and acid washed. In permeabilized and fixed HCAEC, VEGFR-2 was detected with an AlexaFluor488-conjugated secondary antibody and is shown in green. c-Src was labeled with AlexaFluor647-conjugated secondary antibody (red) and nuclei with DAPI (blue). (B) Bar graphs show image analysis for colocalization events using the NIH Image J plugin (as described in Materials and methods). The graphs present the number of colocalization events normalized for the number of total VEGFR-2–positive immunofluorescence signals. Values are the mean of three experiments ± S.E.M., each containing numbers obtained from five random fields. *p<0.05 was considered statistically significant.
Figure 5
Figure 5. Proposed model: thiol oxidation may help propagate signal transduction from VEGFR-2 to downstream c-Src.
(A) VEGF activation of VEGFR-2 and downstream PLCγ-1-ERK1/2 signaling pathway appears to be independent of ROS levels in ECs. (B) VEGF induces NADPH oxidase-derived ROS, which in turn oxidizes VEGFR-2 and c-Src. Thiol oxidation of these two tyrosine kinases appears to correlate with VEGF-induced activation of c-Src, and also with the sub-cellular colocalization and interaction between VEGFR-2 and c-Src. Dependence of VEGF-induced thiol oxidation and activation of c-Src on NADPH oxidase-derived ROS render downstream activation of PI3K-Akt redox-sensitive in HCAEC. In this model, VEGFR-2 and/or c-Src act as endothelial redox-sensors that determine whether downstream PI3K-Akt signaling pathway should be activated or not.

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