key: cord-0705182-yxyvbfq2
authors: Tang, Sihui; Jiang, Jianjun; Zhang, Na; Sun, Juan; Sun, Gengyun
title: Tumor necrosis factor-α requires Ezrin to regulate the cytoskeleton and cause pulmonary microvascular endothelial barrier damage
date: 2020-09-30
journal: Microvasc Res
DOI: 10.1016/j.mvr.2020.104093
sha: a0e1bdf4a29613aba7f8406e318e632ea229336b
doc_id: 705182
cord_uid: yxyvbfq2

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 (0,1,10,100ng / 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 WesternBlot 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 effectof 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.

ARDS is a rapidly progressive disease occurring in critically ill patients.The most common causes of ARDS are pneumonia, aspiration of liquid contents, septicemia, acute liver failure, and acute pancreatitis, which directly or indirectly induce lung injury [1] [2] [3] . At the same time, the exact pathogenies still remain unclear. Some studies have suggested that the accumulation of neutrophils and proinflammatory cytokines around pulmonary microvascular endothelial cells (PMVECs) may lead to the destruction of intercellular connections, microtubule activation, and actin cytoskeleton remodeling, cell contraction and formation of gaps, which then allows a large amount of protein and fluid to enter the pulmonary interstitium and alveolar cavity, resulting in severe hypoxic respiratory failure [4] [5] [6] . Therefore, it is of potential and far-reaching clinical significance to study the molecular mechanisms of inflammatory injury of PMVECs barrier.

Ezrin-Radixin-Moesin (ERM) protein family is a group of membrane-related proteins with high homology, composed of three functional domains: the N-terminal FERM domain[band four-point-one, ezrin, radixin, moesin homology domains], the extended, curly helix domain, and the short C-terminal domain [7] .

As a member of ERM, Ezrin exists in two conformational states. At rest, it exists in the form of folded inactivation. When stimulated by an effective signal, the N-terminal and C-terminal of the fold separate from each other, resulting in the phosphorylation of threonine at site 567th in the molecular structure, and the connection with phosphatidylinositol diphosphate [phosphtidyli-nositol (4,5)-bisphosphate, PIP2] on the surface of N-terminal cell membrane leads to protein, cortactin) has a crucial role in actin cytoskeleton regulation. A large number of studies have confirmed that the increased distribution of F-actin and Cortactin in the cortex often indicates cell expansion, enhanced cell-to-cell junction, and decreased monolayer permeability of endothelial cells [11，12] .It has also been confirmed that as a member of the RhoGTPase family, ras-related C3 botulinum toxin substrate 1 (Rac1) is essential in stabilizing the actin cytoskeleton and maintaining microvascular barrier function [13， 14] . Rac1 regulates the function of the cytoskeleton and intercellular junction mainly by promoting the formation of actin fiber bundles and the remodeling of junction complexes in the cortex [15，16] . Nevertheless, the molecular mechanism of Rac1 involved in the regulation of PMVECs permeability under inflammatory conditions still remains unclear.

TNF-α is a component that promotes and drives the inflammation of many lung diseases and conditions, including ARDS [17，18] . Our previous studies have found that TNF-α can induce F-actin rearrangement in PMVECs, increasing cell permeability [11] . However, the specific molecular mechanism of TNF-α destroying PMVECs barrier function is still not clear. We speculated that Ezrin and Rac1 might have a role in TNF-α-related pathways, regulating the permeability of PMVECs. To address this hypothesis, we used TNF-α to treat PMVECs, detected the effects of Ezrin and Rac1, respectively,and determined the mechanism of Ezrin and Rac1 in TNF-α mediated endothelial barrier destruction.

Rat TNF-α was purchased from PeproTech (New Jersey, USA). Ezrin and J o u r n a l P r e -p r o o f were purchased from HyClone Laboratory (Salt Lake City, USA).

Primary RPMVECs were isolated and cultured according to the mature techniques in our laboratory [19，20] .A total of 30 healthy male SD rats weighing 90-120g were purchased from the Animal Experimental Center of Anhui Medical University. After intraperitoneal injection of pentobarbital sodium to full anesthesia, the rats were sacrificed, the pleura was removed, and the subpleural lung tissue without large blood vessels was separated and cut into small pieces, and placed in a culture flask.DMEM containing 15% FBS was added to the culture flask, which was placed in the incubator (37 °C, 5%CO2) for 60 h.The tissue mass was removed, and the culture medium was changed every 3 days. All 

Cell viability was analyzed using a CCK-8 kit. After cell counting, PMVECs (1×10 4 cells per well) were plated in 96-well plate and incubated overnight in a humidified atmosphere containing 5%CO 2 /95% air at 37ºC. Cells were then exposed to gradually increased concentration of TNF-α (0, 1, 10, 100ng / ml) for 2h, after which 10μl of sterile CCK8 was added to each well, and cells were incubated for another 3h at 37°C. The absorbance of each group was detected at 450nm byMicroplate Reader (Enspire, PerkinElmer, USA), and a column chart was drawn to reflect the activity of cells.

Lentiviruses containing shRNA-Rac1 (shRac1) and shRNA-Ezrin (shEzrin) were purchased from GenePharma Pharmaceutical Technology Co.Ltd. (Shanghai, China) and was used to regulate the expression of Rac1 and Ezrin. PMVECs, grown to more than 90%fusion in a culture flask, were inoculated in a six-well plate. On the third day of culture, the medium was replaced with the medium containing 15% fetal bovine serum, 100uL lentivirus, and 5mg/mLpolybrene. Then, 48-72 hours after transfection, the protein was photographed by a fluorescence microscope, and the protein was collected for Western blot analysis to verify the silencing effect of Rac1 and Ezrin.

RPMVECs (2×10 5 /cm 2 ) were inoculated on Matrigel-coatedtranswell polyester membranes (Costar, USA). The cells were cultured for more than 48 hours until 

When the TEER value was stable, and confluency with stable junctions was observed under the microscope, the cells were starved with DMEM containing 1% fetal bovine serum for 12 hours and rinsed with PBS for 3 times. According to the experimental scheme, different stimulating factors act on the cells. The concentration of FITC-BSA 0.1ml in the upper chamber was 0.1mg/ml, and the same concentration of BSA0.6ml was added in the lower chamber. After incubation in a 5%CO2 incubator at 37℃for 2 hours, 0.1ml samples were collected from the lower chamber and added into 96-well plates. The excitation wavelength of the luciferase-labeling instrument was set to 488nm, and the fluorescence intensity of the absorbed samples was measured.

The active form of intracellular Rac1 (Rac1-GTP) was determined by a commercial kit. After stimulation with TNF-α, the PMVECs were lysed with the lysis buffer, and the immobilized PAK1-PBD was used to capture Rac1-GTP by pull-down experiment. The levels of Rac1-GTP and total Rac1 were analyzed by Western blot.

RPMVECs (1×10 4 ) were treated with TNF-α or O-Me-cAMP for a certain period. Cells were then fixed with 4% paraformaldehyde for 15 min, blocked with 0.3% Tritonx 100 and 5% goat serum in PBS for 1 hour, and incubated with 1:200 anti-Cortactin antibody at 4˚C overnight. Consequently, cells were stained with rhodamine-Phalloidin (1:400) and cy3 (1:400) combined with F-actin and cortactin, respectively. The nucleus was stained with 0.5mg/mL DAPI for 30min.

All images were obtained under a fluorescence microscope (Axio Observer3, ZEISS, Germany).

PMVECs (2x10 6 cells/cm 2 ) were collected, and the protein was extracted and quantified according to the manufacturer's instructions. A 15%, 12% separation gel, and 5% concentrated gel were prepared for protein loading and electrophoresis. The target protein was then transferred to the PVDF membrane by semi-dry method, sealed for 2 hours, incubated with primary antibody at 4 degrees overnight, and with the secondary antibody for 1 hour after Tris-buffered saline with Tween 20 (TBST)washing. After washing the PVDF film again with TBST, the gel imaging system (AI600RGB, GE, USA) was used to develop the film, and the gray value of the band was analyzed by ImageJ software.

SPSS20.0 was used to analyze the data. The results were expressed by the mean ±standard deviation (x±s). The pairwise comparison of multiple variables was analyzed by one-way ANOVA, and the independent sample t-test was used to compare the variables between the two groups. A p<0.05 was considered to be statistically significant.

In the present study, PMVECs were identified by binding with lectin from BSI (FITC-BSI) staining and cell purity was more than 90% (Supplemental Figure   S1 ). We confirmed previous results that TNF-α reduced the TEER of PMVECs in a time-dependent and dose-dependent manner without affecting cell viability( Figure 1A) .Next, we measured the TEER of cells in each group under specific intervention conditions. Compared to the control group, 1-100ng/mL J o u r n a l P r e -p r o o f Journal Pre-proof TNF-α decreased the TEER in PMVECs in a time-dependent manner ( Figure 1B and C). Furthermore, FITC-BSA flux increased in a concentration-dependent manner ( Figure 1D) . These data indicated that the TNF-α could damage the barrier of and increase the permeability of PMVECs. The most significant effect was observed at 100ng/mL TNF-α, which was selected for further experiments. 

To elucidate the effect of TNF-α on phosphorylation of Ezrin at its critical J o u r n a l P r e -p r o o f COOH-terminal threonine, phospho-specific Ezrin antibody (Thr567) was utilized to evaluate threonine phosphorylation of Ezrin by Western Blot.

Treatment with 100 ng/mL TNF-α induced a significant increase in Ezrin phosphorylation in a time-dependent manner without affecting the total Ezrin expression, which increased after 15 min, reached maximum levels by 2h, and remained elevated for at least 8h (Figure 2) . 

To explore the signaling mechanism of Ezrin phosphorylation, we used Rac1-specific shRNA (shRac1). The results showed that the threonine phosphorylation of Ezrin was increased after transfecting PMVECs with shRac1 alone. ShRac1 combined with TNF-αsignificantly increased Ezrin threonine phosphorylation in PMVECs (Figure 4 ). The following experiments were used to determine the role of Ezrin in TNF-α-induced RPMVECs barrier destruction. ShRNA targeting Ezrin was used to inhibit the expression of Ezrin (Figure 5A and 5B) but not the expression of Moesin or Radixin (Figure 5C and 5D) , after which changes in TEER and FITC-BSA permeability of cells were measured. The results showed that Ezrin silencing could partially recover the decrease of TEER and the increase of FITC-BSA permeability induced by TNF-α exposure (Figure 5E and 5F) , which indicated that Ezrin silencing could reduce the damage of PMVECs barrier induced by TNF-α.

The distribution of F-actin in the cortical region and cortactin on the J o u r n a l P r e -p r o o f membrane is one of the key factors for maintaining the endothelial barrier. Next, we examined the effect of Ezrin on cytoskeleton rearrangement. As shown in Figure 6 , after transfecting cells with control-shRNA or shEzrin, F-actin was evenly arranged and distributed all over the cell (more on the cell membrane), while cortactin was mainly found in the cytoplasm. When PMVECs were exposed to TNF-α, a large number of F-actin gathered and formed stress fibers in the cytoplasm, and the level of cortactin on the cell membrane decreased.

PMVECs pretreated with shEzrin reversed F-actin rearrangement and cortactin redistribution induced by TNF-α exposure (Figure 7) . These results were consistent with the immunofluorescence analysis.Therefore, we inferred that Ezrin was essential in the destruction of the PMVEC barrier mediated by TNF-α partly by inducing cytoskeleton remodeling. 

Rac1 is an important regulator of actin cytoskeleton dynamics that has a key role in maintaining endothelial integrity. After pre-incubation with O-me-cAMP, a specific agonist of Rac1, we found that O-me-cAMP partially restored the decrease of TEER and the increase of FITC-BSA permeability induced by TNF-α ( Figure 8A and 8B) , and decreased the vascular permeability induced by TNF-α.

Moreover, a Western blot analysis indicated that Ezrin inhibition promotes the expression of active rac1 and significantly reduces the decrease of rac1-GTP expression induced by TNF-α exposure (Figure 8C and 8D) . These results suggested that both TNF-α and Ezrin could inhibit the activity of Rac1. 

To confirm whether Rac1 activity was directly related to Ezrin-induced barrier injury, we co-transfected RPMVECs with shEzrin and shRac1, then treated with TNF-α, and finally evaluated changes in cell permeability. As shown in Figure 9 , 

As a typical clinical emergency and critical illness, ARDS has a high mortality rate and is still lacks effective treatment. The lung injury in ARDS is mainly caused by the dysfunction of RPMVECs, which are triggered by systemic inflammation [4，21] . Therefore, it is crucial to explore the signaling pathways related to ARDS, which, in turn, may affect the function of RPMVECs. This study described the effects of Ezrin and Rac1 on the permeability of PMVEC J o u r n a l P r e -p r o o f monolayers in an inflammatory environment mediated by TNF-α. We found that TNF-α increases PMVEC permeability in a doseand time-dependent manner. In addition, the intracellular Ezrin threonine phosphorylation increased with the prolongation of TNF-α stimulation time, thus suggesting that Ezrin has a vital role in mediating TNF-α-induced endothelial barrier dysfunction. By silencing Rac1 and Ezrin with shRNA, our data suggested that Rac1 and Ezrin could regulate each other and participate in mediating TNF-α PMVECs monolayer permeability increase by regulating the cytoskeleton.

TNF-α is an essential inflammatory modulator involved in a variety of pathological processes such as inducing the accumulation of inflammatory cells, stimulating the production of inflammatory mediators, proliferation, and differentiation of injury and infected sites, as well as airway hyperreactivity and tissue remodeling [5，22，23] . In the present study, we found that TNF-α induced is regulated by the phosphorylation of multiple kinases in multiple domains [24] .

The most common Ezrin phosphorylation site is threonine 567 in the C-terminal domain [25] . Our results revealed that TNF-α stimulation increases the intracellular p-Ezrin (Thr567) in a time-dependent manner but does not affect the total intracellular Ezrin expression. In lung diseases, Ezrin has been confirmed to be related to the pathogenesis of asthma [26] . cAMP/PKA pathway induces contraction of airway smooth muscle cells and the production of cytokines by J o u r n a l P r e -p r o o f phosphorylating Ezrin. In the early stage of asthma, epidermal growth factor (EGF) can induce the phosphorylation of Ezrin, which connects CD44 and cortical actin cytoskeleton, and participates in the repair of bronchial epithelium [27] . Currently, there are only few studies on lung injury and pulmonary infection. To further examine the connection of Ezrin and TNF-α stimulation in vitro, we transfected the PMVECs with shEzerin vector. Our finding revealed that the inhibition of Ezrin could partially alleviate the damage of the PMVEC barrier caused by TNF-α.Therefore, it can be inferred that activated Ezrin was involved in barrier dysfunction mediated by TNF-α.

Rac1, Cdc42, and Rap1 can help to maintain and stabilize the function of the microvascular endothelial cell barrier [28，29] . In the past few years, the role of rac1 in the assembly of endothelial junctions and the increase of its activity in the process of junction formation have attracted a lot of attention [30] . Rac1 regulates cytoskeletal actin rearrangement through a variety of mechanisms [31] [32] [33] . In this experiment, we used TNF-α to stimulate RPMVECs, and then measured the expression of Rac1 at different time points (0, 0.5, 1, 1.5, 2, and 3h) using Western blot. TNF-α had no effect on the expression of total Rac1 protein, but the content of active Rac1 (Rac1-GTP) decreased gradually with time. By measuring the permeability of PMVEC monolayers pre-incubated with O-me-cAMP, a specific agonist of Rac1, we found that the up-regulation of Rac1 activity could reduce PMVEC hyperpermeability induced by TNF-α. Of note, O-me-cAMP was also reported to activate Rac1 via Epac/Rap1 signalling [34] . In the future, more refined studies are required to understand the complex interaction between Rac1, Epac and Rap1. ShEzrin increased the expression of active Rac1 and significantly reduced the decrease of intracellular Rac1 activity induced by TNF-α. When shRac1 was added to cells, the protective effect of shEzrin was weakened. Wójciak-Stothard et al found that in human umbilical veinendothelial cells (HUVECs) [35] , TNF-α can affect both RhoA and Rac1.

Moreover, in HUVECs, TNF-α enhances hyperpermeability of HUVECs infected Listeria monocytogenes via RhoA signaling pathway [36] . In this study, we showed that RhoA was activated by both TNF-α and sh-Rac1 in PMVEC (Supplemental Figure S2 ). More interestingly, shRac1 led to rearrangement of the distribution of F-actin and cortactin in PMVEC (Supplemental Figure S3) , suggesting that RhoA signaling pathway may also be involved in regulating the increase of cell permeability induced by TNF-α. Besides founding that shEzrin activated Rac1, we also observed a significant increase in phosphorylated Ezrin induced by shRac1, i.e. Rac1 was not only the downstream signal target of Ezrin, but also the upstream regulatory factor of phosphorylation in the process of endothelial barrier destruction induced by TNF-α.

McKenzie et al found that in human PMVEC, TNF-α can induce the activation of PKC to phosphorylate the C-terminal threonine of Ezrin, resulting in increased endothelial permeability [7] . Our study further revealed that TNF-α could directly promote Ezrin phosphonic acid and regulate endothelial barrier function by regulating the activity of Rac1. Adyshev et al suggested that ERM differentially regulates S1P-induced changes in the cytoskeleton and permeability of lung EC cells, among which Radixin has the most significant role [37] . Moesin and total ERM have a prominent role in thrombin-induced cytoskeleton rearrangement [38] . Our results confirmed that Ezrin, as a member of ERM, is important for the cytoskeleton rearrangement of PMVEC cells induced by TNF-α. Moreover, Pujuguet et al found that phosphorylated Ezrin activated Rac1 can further activate the Rho signal pathway and regulate the expression of E-cadherin in the cytoplasm and cell membrane [39] . This was not consistent with our results, according to which phosphorylated Ezrin inhibited Rac1 activation, likely because of different cell models.

The present study has a few limitations. First, all the experiments were conducted in vitro using rat PMVECs. Future studies should evaluate the effects J o u r n a l P r e -p r o o f of Ezrin silencing on PMVECs in animal models, and determine the effects of permeability and cytoskeleton remodeling in inflammatory human PMVECs.

Secondly, the specific mechanism of p-Ezrin in regulating the increase of cell permeability induced by TNF-α is still not fully clear, especially the mechanism of the Ezrin-related signal transduction pathway. Also, potential cooperation between related signal transduction pathways needs to be further investigated, especially the crosstalk effect of ICAM-2 and Rac1-Ezrin signaling [40] .

To sum up, our preliminary data suggest that Ezrin participates in the experimental phenomenon of TNF-α-induced increase of PMVEC permeability by regulating Rac1 activity and the process of regulating the subcellular structure of the cytoskeleton. ShEzrin did not completely reverse the barrier damage caused by TNF-α, which indicated that there are other mechanisms involved in the regulation of the downstream of the TNF-α pathway. It is worth mentioning that the activity of Rac1 can also negatively regulate the phosphorylation of Ezrin. These results enhance our understanding of the molecular mechanism of ARDS and the function of the pulmonary microvascular barrier.

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This study was supported by National Natural Science Foundation of China (No. 81370170 and 81770081).

The authors declare that there is no conflict of interest regarding the publication of this paper.

This paper has not been published elsewhere in whole or in part. All authors have read and approved the content, and agree to submit it for consideration for publication in your journal. There are no conflicts of interest involved in the article.J o u r n a l P r e -p r o o f