Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability.
Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability.
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PMVEC 中炎症免疫细胞因子 TNF-α 通过 FAK/RhoA 信号通路调节 Ezrin 蛋白激活
DOI：
10.3389/fphar.2021.676817
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发表时间：
2021
影响因子：
5.6
通讯作者：
Sun G
中科院分区：
文献类型：
--
作者：
Zhou Q;Jiang J;Chen G;Qian C;Sun G
背景：急性呼吸窘迫综合征（ARDS）的重要发病机制之一是高炎症免疫反应引起的肺微血管内皮屏障功能障碍。然而，这种肺微血管内皮细胞（PMVECs）失衡的潜在机制尚不清楚。目的：探讨急性呼吸窘迫综合征（ARDS）中炎症免疫细胞因子诱导内皮屏障功能障碍的分子机制，寻找治疗该综合征的靶点。方法：培养大鼠pmvec形成单层。采用免疫荧光、流式细胞术、Western blotting检测磷酸化Ezrin蛋白和Ezrin蛋白的分布及表达水平。利用异硫氰酸荧光素（FITC）标记的牛血清白蛋白（BSA）的跨内皮电阻（TER）和跨内皮通量来测量细胞单层的通透性。利用Ezrin短发夹RNA （shRNA）和Ezrin 567位点苏氨酸突变体（EzrinT567A）分别检测Ezrin蛋白和磷酸化Ezrin蛋白在肿瘤坏死因子-α (TNF-α)诱导的内皮反应中的作用。通过抑制剂和RhoA/FAK shRNA在TNF-α刺激的大鼠pmvec中评估FAK和Ras同源基因家族成员A （RhoA）信号通路的功能。采用Western blotting或pull-down assay + Western blotting评估FAK和RhoA的活化情况。结果：TNF-α作用后，小鼠TER水平降低，Ezrin蛋白磷酸化水平升高，且呈时间和剂量依赖性。磷酸化的Ezrin蛋白主要定位于细胞周围，导致丝状肌动蛋白（F-actin）重排，随后TER显著降低，FITC-BSA通量增加。此外，FAK和RhoA信号通路在Ezrin蛋白磷酸化过程中是必需的，前者正调控后者。结论：TNF-α通过FAK/RhoA信号通路诱导Ezrin蛋白磷酸化，导致pmvec内皮细胞高通透性。
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 (EzrinT567A) 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.
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影响因子：
4.8
作者：
Adyshev DM;Moldobaeva NK;Elangovan VR;Garcia JG;Dudek SM
通讯作者：
Dudek SM
影响因子：
20.1
作者：
Naikawadi RP;Cheng N;Vogel SM;Qian F;Wu D;Malik AB;Ye RD
通讯作者：
Ye RD
磷酸肌醇的结合和磷酸化在ezrin的激活机理中依次起作用。
影响因子：
7.8
作者：
Fievet, BT;Gautreau, A;Roy, C;Del Maestro, L;Mangeat, P;Louvard, D;Arpin, M
通讯作者：
Arpin, M
DOI：
10.1002/path.4911
发表时间：
2017-07
期刊：
The Journal of pathology
影响因子：
--
作者：
Alexopoulou AN;Lees DM;Bodrug N;Lechertier T;Fernandez I;D'Amico G;Dukinfield M;Batista S;Tavora B;Serrels B;Hodivala-Dilke K
通讯作者：
Hodivala-Dilke K
影响因子：
4.8
作者：
Ben-Aissa, Khadija;Patino-Lopez, Genaro;Shaw, Stephen
通讯作者：
Shaw, Stephen