Abstract
Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.
Highlights
(VEGF)2 is essential for many angiogenic processes both in normal and pathological conditions [3]
In contrast to the reduced Akt and endothelial nitric-oxide synthase (eNOS) phosphorylation by VE- stimulated c-Src activation and downstream signaling to Akt, cadherin small interference RNA (siRNA), there was an increase of Vascular endothelial growth factor (VEGF)-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) eNOS, and ERK1/2 is specific, because siRNA silencing of phosphorylation in VE-cadherin siRNA-treated Human umbilical vein endothelial cells (HUVECs) com- platelet-endothelial cell adhesion molecule, another paracellupared with control cells (Fig. 2, C and D)
We found that VEGF stimulated C-terminal Src kinase (Csk) dissociation with VE-cadherin, resulting in the activation of c-Src
Summary
Reagents and Antibodies—VEGF was from R&D Systems, Inc. (Minneapolis, MN). U73122, LY294002, wortmannin, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo{3,4-d}pyrimidine (PP2) were from Calbiochem. Immunofluorescence—For analysis of the VE-cadherin, Csk, and phospho-Src Tyr-416 localization in monolayer endothelial cells, BAECs or HUVECs were fixed with 3.7% formaldehyde in phosphate-buffered saline after the treatment of VEGF [31]. VEGF stimulated increased Src Tyr-416 phosphorylation in control siRNA-treated cells in a time-dependent manner, which is similar to untreated cells (not shown). VEGF-evoked c-Src Tyr-416 phosphorylation was decreased markedly in VE-cadherin siRNA-treated cells (Fig. 1, A and B). VEGFR2 tyrosine phosphorylation in response to VEGF (Fig. 1C) These results imply that VE-cadherin plays a critical role in c-Src activation by VEGF in ECs. VE-cadherin Differentially Modulates Akt and ERK1/2 Activation by VEGF—To further determine the role of VE-cadherin in VEGF signaling, we examined the effects of silencing VE-cadherin by siRNA. On VEGF-induced activation of ERK1/2, Akt, and eNOS (a substrate of Akt [32]), which have been shown to play pivotal roles in endothelial cell proliferation, survival, migration, and permeability [33]
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