Tiam1 and Rac1 Are Required for Platelet-activating Factor-induced Endothelial Junctional Disassembly and Increase in Vascular Permeability

Ivana I Knezevic,Sanda A Predescu,Radu F Neamu,Matvey S Gorovoy,Nebojsa M Knezevic,Cordus Easington,Asrar B Malik,Dan N Predescu

doi:10.1074/jbc.m808958200

Abstract

It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.

Highlights

The structural integrity of the endothelial cells (ECs) along with their proper functionality are the two most important factors controlling the tightness of the endothelial barrier
We found that the opening of endothelial barrier and the increased vascular leakiness induced by platelet-activating factor (PAF) are the result of a shift in actin pools without involvement of EC contraction, followed by a redistribution of tight junctional associated protein ZO-1 and adherens junctional protein VE-cadherin
It was rationalized that a functional endothelial barrier is the result of the equilibrium between an intermediate contractile status of ECs and tightly organized interendothelial junctions (IEJs)

Summary

Introduction

The structural integrity of the ECs along with their proper functionality are the two most important factors controlling the tightness of the endothelial barrier. Similar to the complexity of actin-interacting proteins found in other cell types, the ECs. Mechanisms of PAF-induced Increase in Vascular Permeability utilize their actin binding proteins to stabilize the endothelial monolayer in order to efficiently function as a selective barrier [11]. C2 toxin increases endothelial permeability by ribosylating monomeric G-actin at Arg-177 [22] This results in the impairment of actin polymerization [23], followed by rounding of ECs [16] and the disruption of junctional integrity. PAF-mediated activation of ECs induced cell migration [32], angiogenesis [7], and vascular hyperpermeability [33] secondary to disassembly of IEJs [34]. Studies have shown that PAF challenge induced endothelial actin cytoskeletal rearrangement [37] and marked vascular leakiness [38]; the signaling pathways have not been elucidated

Methods

Results

Conclusion