Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions

Wei Zhang,Charu Rajput,Pascal Yazbeck,Monica Smith,Nebojsa Knezevic,Dolly Mehta,Alejandra Chavez,Vidisha Kini,Tracy Schmidt,Yulia Komarova

doi:10.1074/jbc.m112.440396

Abstract

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

Highlights

Mechanisms linking cortical actin with adherens junctions (AJs) required for forming restrictive endothelial barrier remain unclear
F, HPAEC plated on gold electrodes were transfected with siSc or siN-WASP, and changes in transendothelial electrical resistance (TER) were determined in real time at the indicated time points
We show that TER remained significantly lower at each time point in N-WASPdepleted Endothelial cells (EC), recapitulating the above findings (Fig. 1C)

Summary

Background

Mechanisms linking cortical actin with adherens junctions (AJs) required for forming restrictive endothelial barrier remain unclear. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. The cortical actin, the actin ring near the plasma membrane, is required for stabilizing AJs (7–10) Edemagenic agonists such as thrombin rapidly induce actin reorganization from cortical actin into actin stress fibers, which induces AJ disruption and interendothelial gap formation, leading to increased endothelial permeability that can result in protein-rich tissue edema and increased transendothelial migration of inflammatory cells (2, 8 –11). FAK and Cdc are known to maintain AJ function in part by interacting with p120-catenin (25–27) It remains unknown whether N-WASP regulates cortical actin formation during alteration of AJ function and contributes to attainment of basal endothelial barrier function. We further show that FAK-mediated N-WASP phosphorylation is the critical step in inducing N-WASP interaction with p120-catenin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION