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

Abstract

Background: One of the important pathogenesis of acute respiratory distress syndrome (ARDS) is the dysfunction of pulmonary microvascular endothelial barrier induced by a hyperinflammatory immune response. However, the potential mechanisms of such an imbalance in pulmonary microvascular endothelial cells (PMVECs) are not yet understood. Purpose: Explore the molecular mechanism of endothelial barrier dysfunction induced by inflammatory immune cytokines in ARDS, and find a therapeutic target for this syndrome. Methods: Rat PMVECs were cultured to form a monolayer. Immunofluorescence, flow cytometry, and Western blotting were selected to detect the distribution and the expression level of phosphorylated Ezrin protein and Ezrin protein. Transendothelial electrical resistance (TER) and transendothelial fluxes of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) were utilized to measure the permeability of the cell monolayer. Ezrin short hairpin RNA (shRNA) and Ezrin 567-site threonine mutant (Ezrin^T567A) were used to examine the role of Ezrin protein and phosphorylated Ezrin protein in endothelial response induced by tumor necrosis factor-alpha (TNF-α), respectively. The function of focal adhesion kinase (FAK) and Ras homolog gene family, member A (RhoA) signaling pathways were estimated by inhibitors and RhoA/FAK shRNA in TNF-α-stimulated rat PMVECs. The activation of FAK and RhoA was assessed by Western blotting or pull-down assay plus Western blotting. Results: The TER was decreased after TNF-α treatment, while the Ezrin protein phosphorylation was increased in a time- and dose-dependent manner. The phosphorylated Ezrin protein was localized primarily at the cell periphery, resulting in filamentous actin (F-actin) rearrangement, followed by a significant decrease in TER and increase in fluxes of FITC-BSA. Moreover, FAK and RhoA signaling pathways were required in the phosphorylation of Ezrin protein, and the former positively regulated the latter. Conclusion: The phosphorylated Ezrin protein was induced by TNF-α via the FAK/RhoA signaling pathway leading to endothelial hyperpermeability in PMVECs.

Keywords: Ezrin protein; acute respiratory distress syndrome; endothelial permeability; inflammatory immune response; molecular mechanism.

FIGURE 1

Effect of Ezrin shRNA on the rearrangement of F-actin and the distribution of threonine phosphorylated Ezrin/Ezrin protein (Thr-pEzrin/Ezrin). (A) For each panel, the images from left to right showed rhodamine fluorescence (red), Alexa Fluor 488 fluorescence (green), cell nuclei stained with DAPI (blue), and overlays of the three images. In control shRNA cells, few stress fibers were observed in the cytoplasm and phosphorylated Ezrin protein was primarily located in the cytoplasm. Ezrin shRNA showed a phenomenon similar to control one. Control shRNA cells exposure to 20 ng/ml TNF-α for 2 h induced F-actin recombination and gather of phosphorylated Ezrin protein to cell periphery. Downregulation of Ezrin protein weakened the F-actin band and induced a decrease of phosphorylated Ezrin protein locating to cell periphery. (B) The distribution of Ezrin protein was not altered by TNF-α while F-actin had a similar trend to (A). Scale bar, 20 μm. (C) The fluorescence intensity of F-actin, phosphorylated Ezrin protein and Ezrin protein was detected via flow cytometry. (D) The mean fluorescence intensity (MFI) from flow cytometry. For each condition, the experiment was repeated three times, all of which had similar observations. Data was presented as mean ± SD. n = 3, **p < 0.01 vs. control group. ^# p < 0.05 between TNF-α + control shRNA and TNF-α + Ezrin shRNA groups.

FIGURE 2

Effect of Ezrin shRNA on the expression level of Thr-pEzrin in TNF-α-induced PMVECs. (A) Ezrin shRNA downregulated the expression level of Ezrin protein. (B) Ezrin protein was not sensitive to TNF-α (20 ng/ml, 2 h) stimulation. Ezrin shRNA alleviate the increase of Thr-pEzrin induced by TNF-α. (C–E) Relative expression levels from the Western blotting. Data were presented as mean ± SD. n = 3, **p < 0.01 vs. control group. ^$ p < 0.05 between 24 h and 48 h groups. ^& p < 0.05 between 48 h and 72 h groups, ^# p < 0.05 between TNF-α + control shRNA and TNF-α + Ezrin shRNA groups.

FIGURE 3

Effect of Ezrin shRNA on endothelial hyperpermeability in TNF-α-induced PMVECs. (A) Effect on TER of PMVECs monolayer. (B) Effect on fluxes of FITC-BSA across PMVECs monolayer. Each bar represented mean ± SD of three independent trials; ^** p < 0.01 vs. control group. ^# p < 0.05 between the TNF-α + control shRNA group and the TNF-α + Ezrin shRNA group.

FIGURE 4

Effect of phosphorylation mutant of Ezrin protein (Ezrin^T567A) on endothelial responses in TNF-α-induced PMVECs. (A) Ezrin^T567A can reverse the localization of Thr-pEzrin and reduce the recombination of F-actin induced by TNF-α. Scale bar, 20 μm. (B) The MFI of F-actin, Thr-pEzrin was calculated via flow cytometry. (C) Effect of Ezrin^T567A on the Thr-pEzrin expression level induced by TNF-α. (D) Ezrin^T567A relieved the decline of TER and increased the fluxes of FITC-BSA induced by TNF-α. For each condition, the experiment was repeated three times, all of which had similar observations. Data were presented as mean ± SD. n = 3, ^* p < 0.05 vs. control group. **p < 0.01 vs. control group. ^# p < 0.05 between TNF-α + Ezrin^WT and TNF-α + Ezrin^T567A groups.

FIGURE 5

Effect of RhoA or FAK inhibitor on the expression level of Thr-pEzrin in TNF-α-induced PMVECs. (A) RhoA inhibitor prevented TNF-α-induced Ezrin phosphorylation. PMVECs were pretreated with either vehicle or 2 μg/ml C3 transferase for 6 h before they were treated with 20 ng/ml TNF-α for 2 h (B) FAK inhibitor alleviated TNF-α–induced Ezrin phosphorylation. PMVECs were pretreated with either vehicle or 1 μM PF-573228 for 0.5 h before they were treated with 20 ng/ml TNF-α for 2 h (C,D) The according expression level of phosphorylated Ezrin protein from the Western blotting. Data were presented as mean ± SD. n = 3, ^* p < 0.05, and **p < 0.01 compared to the control group. ^# p < 0.05 between the TNF-α group and the TNF-α + inhibitor group.

FIGURE 6

Effect of RhoA or FAK inhibitor on endothelial hyperpermeability in TNF-α-induced PMVECs. (A,B) RhoA or FAK inhibitor protected the decrease of TER treated with TNF-α. (C, D) RhoA or FAK inhibitor reduced the fluxes of FITC-BSA. Data were presented as mean ± SD. n = 3, **p < 0.01 compared to the control group. ^# p < 0.05 between the TNF-α group and the TNF-α + inhibitor group.

FIGURE 7

Effect of RhoA shRNA or FAK shRNA on the rearrangement of F-actin and the distribution of Thr-pEzrin/Ezrin. (A,B) The TNF-α responses were prevented remarkably via RhoA shRNA and FAK shRNA treatment, respectively (C,D) There was no change in Ezrin protein under the same stimulation. Scale bar, 20 μm. (E,F) The fluorescence intensity of F-actin, Thr-pEzrin/Ezrin was detected via flow cytometry. (G,H) MFI from flow cytometry. All images were representative of three independent experiments with similar observations. Data were presented as mean ± SD. n = 3, **p < 0.01 vs. control group. ^# p < 0.05 between TNF-α + control shRNA and TNF-α + RhoA/FAK shRNA groups.

FIGURE 8

Effect of RhoA shRNA or FAK shRNA on the expression level of Thr-pEzrin in TNF-α-induced PMVECs. (A) RhoA shRNA downregulated the expression level of RhoA. (B) After FAK shRNA treatment, the expression level of FAK decreased. (C,D) The according expression levels from the Western blotting (E,F) Pretreatment with RhoA shRNA prevented TNF-α–induced Ezrin phosphorylation and so can FAK shRNA. (G,H) The according expression levels from the Western blotting. Data were presented as the mean ± SD. n = 3, **p < 0.01 compared to the control group. ^$ p < 0.05 between 24 h and 48 h groups. ^& p < 0.05 between 48 h and 72 h groups, ^# p < 0.05 between TNF-α + control shRNA and TNF-α + RhoA/FAK shRNA groups.

FIGURE 9

Effect of RhoA shRNA or FAK shRNA on endothelial hyperpermeability in TNF-α-induced PMVECs. (A,B) RhoA or FAK shRNA protected the decrease of TER treated with TNF-α. (C,D) RhoA or FAK shRNA decreased the fluxes of FITC-BSA. Data were presented as mean ± SD. n = 3, ^* p < 0.05 compared to the control group. **p < 0.01 compared to the control group. ^# p < 0.05 between TNF-α + control shRNA and TNF-α + RhoA/FAK shRNA groups.

FIGURE 10

The FAK signaling pathway can upregulate the activity of RhoA in TNF-α-induced PMVECs. (A) RhoA shRNA cannot block FAK phosphorylation (p-FAK) induced by TNF-α. (B) FAK shRNA can inhibit RhoA activity (RhoA-GTP). (C,D) Relative expression levels from the Western blotting. Data were presented as mean ± SD. n = 3, ^* p < 0.05, **p < 0.01 compared to the control group.