Abstract
Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE-cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin-blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased production of reactive oxygen species. Moreover, VE-cadherin-associated β-catenin is tyrosine-phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE-cadherin homotypic adhesion. Conversely, the inhibition of Pyk2 activity in endothelial cells by the expression of CRNK (CADTK/CAKβ-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in β-catenin tyrosine phosphorylation but also prevents the loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS through the tyrosine phosphorylation of β-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity.
Highlights
VE-cadherin is linked to the actin cytoskeleton via the armadillo family members - and ␥-catenin that bind the actinbinding protein ␣-catenin [3, 4]
We found that the loss of VE-cadherin function activates the small GTPase Rac1 and increases production of reactive oxygen species (ROS), which subsequently leads to the loss of cell-cell adhesion
This reduced cell-cell adhesion is accompanied by increased tyrosine phosphorylation of -catenin, which depends on the activation of proline-rich tyrosine kinase 2 (Pyk2)
Summary
VE-cadherin is linked to the actin cytoskeleton via the armadillo family members - and ␥-catenin that bind the actinbinding protein ␣-catenin [3, 4]. The specific association of VE-protein tyrosine phosphatase with VE-cadherin and SHP-2 with -catenin provides further evidence that tyrosine phosphorylation of the VE-cadherincatenin complex is important for the regulation of endothelial cell-cell adhesion. We found that the loss of VE-cadherin function activates the small GTPase Rac and increases production of ROS, which subsequently leads to the loss of cell-cell adhesion. This reduced cell-cell adhesion is accompanied by increased tyrosine phosphorylation of -catenin, which depends on the activation of Pyk. This reduced cell-cell adhesion is accompanied by increased tyrosine phosphorylation of -catenin, which depends on the activation of Pyk2 Together, these data provide novel information on the role of Pyk in the regulation of VE-cadherin-based endothelial cell-cell adhesion and endothelial integrity
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