Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction
Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction
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DOI：
10.1016/j.mvr.2003.09.007
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发表时间：
2004-01-01
影响因子：
3.1
通讯作者：
Verin, AD
中科院分区：
文献类型：
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作者：
Birukova, AA;Smurova, K;Verin, AD
凝血酶诱导的肺内皮单层屏障功能障碍与剧烈的细胞骨架重组、肌动球蛋白收缩激活和间隙形成有关。调节肌球蛋白轻链（MLC）的磷酸化是内皮细胞（EC）收缩和屏障功能障碍的关键机制，这是由Ca2+/钙调素依赖性MLC激酶（MLCK）和rho相关激酶（rho激酶）触发的。MLCK在EC屏障调节中的作用已被先前描述；然而，在凝血酶诱导的肺EC功能障碍中，rho介导的途径尚未精确表征。在这里，我们证明凝血酶诱导的跨内皮电阻（TER）下降表明EC屏障功能障碍是人类和牛肺内皮普遍存在的，并且涉及膜易位和小GTPase Rho及其下游靶点Rho激酶的直接激活。瞬时Rho膜易位与上游Rho激活剂鸟苷核苷酸交换因子p115-RhoGEF易位一致。Rho介导下游靶点的激活，Rho激酶诱导EC MLC磷酸酶（MYPT1）在Thr（686）和Thr（850）位点的磷酸化，导致MYPT1失活、二磷酸MLC积累、肌动蛋白重塑和细胞收缩。特异性rho激酶抑制剂Y27632可消除MYPT1磷酸化、MLC磷酸化，显著减弱应激纤维形成和凝血酶诱导的TER降低。此外，Rho和Rho激酶的显性阴性表达消除了凝血素诱导的应激纤维形成和MLC磷酸化。我们的数据提供了肺EC中Rho介导的信号转导的全面分析，证明鸟苷核苷酸交换因子p115-RhoGEF参与凝血酶介导的Rho调节，并表明Rho， Rho激酶和MYPT1是预防凝血酶诱导的EC屏障破坏和急性肺损伤相关肺水肿的潜在药理学和基因治疗靶点。(C) 2003 Elsevier Inc.版权所有。
Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca2+/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr(686) and Thr(850), resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury. (C) 2003 Elsevier Inc. All rights reserved.