Tumor necrosis factor-α requires Ezrin to regulate the cytoskeleton and cause pulmonary microvascular endothelial barrier damage

Abstract

Acute respiratory distress syndrome (ARDS) is a rapidly progressive disease with unknown pathogenesis. Damage of pulmonary microvascular endothelial cells (PMVECs) caused by inflammatory storm caused by cytokines such as TNF-α is the potential pathogenesis of ARDS. In this study, we examined the role of ezrin and Rac1 in TNF-α-related pathways, which regulates the permeability of PMVECs. Primary rat pulmonary microvascular endothelial cells (RPMVECs) were isolated and cultured. RPMVECs were treated with rat TNF-α (0, 1, 10, 100 ng/ml), and the cell activity of each group was measured using a CCK8 kit. The integrity of endothelial barrier was measured by transendothelial resistance (TEER) and FITC-BSA flux across RPMVECs membranes. Pulldown assay and Western blot was used to detect the activity of RAS-associated C3 botulinum toxin substrate 1 (Rac1) and Ezrin phosphorylation. Short hairpin RNA (shRNA) targeting ezrin and Rac1 was utilized to evaluate the effect of RPMVECs permeability and related pathway. The effects of ezrin and Rac1 on cytoskeleton were confirmed by immunofluorescence. Our results revealed that active Rac1 was essential for protecting the RPMVEC barrier stimulated by TNF-α, while active ezrin could partially destroy the PMVEC barrier by reducing Rac1 activity and regulating the subcellular structure of the cytoskeleton. These findings may be used to create new therapeutic strategies for targeting Rac1 in the treatment of ARDS.

Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability

“…As a result, a similar trend to that in our previous observations stimulated by 100 ng/ml TNF-α was observed (Tang et al, 2021).…”

“…In this study, we systematically investigated the role of Ezrin protein on endothelial permeability in TNF-α-stimulated PMVECs. Moreover, FAK, RhoA, or their combination was considered as the required signal mechanism, which differed from the previous works (Koss et al, 2006;Shao et al, 2013;Fei et al, 2019;Tang et al, 2021). The process can be demonstrated as follows.…”

“…To date, few studies have specifically addressed the involvement of Ezrin protein alone in the regulation of PMVEC monolayer permeability. We previously detected the expression level of phosphorylated Ezrin protein and found that it interacts with Rac1 signaling pathway to regulate PMVECs permeability ( Tang et al, 2021 ). In this study, we intend to further synchronously observe the effects of the distribution and expression level of phosphorylated Ezrin protein on F-actin and endothelial permeability in TNF-α-stimulated PMVECs, and explore whether this process can be realized via RhoA and FAK signaling pathways, which, to our best knowledge, has not been reported.…”

The ROCK-Ezrin Signaling Pathway Mediates LPS-Induced Cytokine Production in A549 Cells

“…ALI and its severe form, ARDS, are potentially fatal complications of sepsis and important causes of high mortality in critically ill patients [21]. Several recent studies have linked ERM to LPS-induced lung injury [4][5][6]. However, the speci c role of ezrin during this process has yet to be elucidated.…”

“…Ezrin/radixin/moesin proteins (ERMs) are members of the protein 4.1 superfamily and function as linkers that connect the actin cytoskeleton to the plasma membrane of cells [3]. Recent studies have reported that ERM phosphorylation results in the loss of endothelial barrier integrity, thus leading to lung in ammation [4,5]. Another research involving an LPS-induced ALI model showed that the introduction of a phospho-null moesin mutant into the mouse lung signi cantly attenuated LPSinduced lung in ammation and vascular leakage, thus suggesting that moesin dephosphorylation protects against lung injury [6].…”