Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin

Abstract

Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia, and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction, and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it.

SH2 Domain-Containing Protein Tyrosine Phosphatase 2 and Focal Adhesion Kinase Protein Interactions Regulate Pulmonary Endothelium Barrier Function

“…Both these agents have been previously observed in vitro to increase endothelial permeability (50)(51)(52). Conversely, in vivo studies using these agents causes varying results.…”

Pathologic Mechanical Stress and Endotoxin Exposure Increases Lung Endothelial Microparticle Shedding

“…The entire list of enriched GO-MF, GO-CC, and GO-biological function (GO-BP) proteins is provided in Excel file 1 in the online supplement. Among the list of the shared proteins, we identified the barrier-regulatory linker moesin as a protein of interest (31). This ERM family member and cytoskeletonassociated protein was consistently present within 18% CS-derived EMPs (present in 5 out of 5 separate mass spectrometry analyses) but not in control EMPs (2 out 5 analyses).…”

“…Moesin is a member of the ERM family of proteins (ezrin, radixin, and moesin) and functions as an actin-binding linker, regulating cellular processes that require membrane cytoskeletal reorganization (49). For example, we recently demonstrated a critical role for ERM proteins in human pulmonary EC cytoskeletal rearrangement and barrier regulation that revealed differential effects of individual ERM proteins on these properties, with moesin promoting increased EC permeability (31,50). Our current data indicate that moesin is markedly enriched in pathologic 18% CS-derived EMPs compared with control EMPs as assessed by Western blot, a finding confirmed in plasma MPs of VILIchallenged mice, indicating a possible role as a novel marker of vascular injury and potential ALI mediator.…”

Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury

“…The putative targets include ezrin/radixin/moesin (ERM) proteins, which may be phosphorylated through p38-dependent mechanisms, 84 but apparently, the role of ERM phosphorylation in EC barrier regulation is agonist-specific. [84][85][86][87] A few studies implicated the involvement of p38 activity in the activation of Rho/Rho kinase pathway and EC barrier dysfunction induced by TGFb and Staphylococcus aureus-derived toxins. 83,88 In contrast, inhibition of p38 has no effect on thrombin-induced MLC phosphorylation, which involves Rho activation.…”