Abstract
Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and beta-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.
Highlights
To limit the activation of other confounding signaling pathways known to be initiated by growth factors or inflammatory mediators, Src family kinases (SFK) were activated by expression of a dominant negative C-terminal Src kinase (DN-Csk), constitutively active Src, or knockdown of Csk
In the studies that follow, we demonstrate that changes in endothelial permeability and the phosphorylation of specific tyrosine residues in VE-cadherin are dependent on the method used to increase active Src
We first wanted to define which tyrosine residues of VE-cadherin were phosphorylated by VEGF in cultures of HDMECs and to determine whether the tyrosine phosphorylation was temporally associated with VEGF-induced changes in the integrity of the endothelial monolayer
Summary
Recent studies have found that tyrosine phosphorylation of VE-cadherin is required for regulating leukocyte trans-endothelial cell migration [16, 17] and that this requires activation of SFKs. In addition, it has been shown that mutation of Tyr-658 or Tyr-731 will attenuate VEGF-induced decreases in barrier function [37]. These results suggest that SFK-dependent tyrosine phosphorylation of VE-cadherin may be required for VEGF-induced loss of barrier function in HDMECs. VEGF may induce the activation of other signaling pathways that may lead to increased permeability.
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