Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: Role of Rho-dependent mechanisms
Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: Role of Rho-dependent mechanisms
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DOI：
10.1002/jcp.20055
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发表时间：
2004-10-01
影响因子：
5.6
通讯作者：
Verin, AD
中科院分区：
文献类型：
--
作者：
Birukova, AA;Smurova, K;Verin, AD
肺内皮细胞单层屏障功能障碍与细胞骨架的剧烈重组、肌动蛋白收缩活性的激活和间隙的形成有关。微管(MT)网络和收缩细胞骨架之间的联系尚未得到充分的研究，然而，临床观察表明，静脉注射抗癌药物和MT抑制剂(如长春花碱)可导致乳腺癌患者突然发生肺水肿。在这项研究中，我们研究了MT和肌动球蛋白细胞骨架之间的串扰，并研究了MT抑制剂诺可达唑(ND)诱导内皮细胞(EC)屏障功能障碍的特定分子机制。我们的结果表明，ND对MT的拆解导致跨内皮细胞电阻(TER)和肌动蛋白细胞骨架重构迅速减少，表明EC屏障功能障碍。这些效应包括ND诱导的Rho GTP酶的激活。Rho介导的下游靶标Rho-Kinase的激活诱导了Rho-Kinase效应分子EC MLC磷酸酶(MYPT1)在Thr(696)和Thr(850)处的磷酸化，导致MYPT1失活。磷酸酶抑制导致二磷酸-MLC的积聚，导致肌球蛋白聚合、应力纤维形成和缝隙形成。Y27632对Rho-Kinase的抑制可阻断ND诱导的MYPT1磷酸化、MLC磷酸化和应激纤维形成。此外，通过MT稳定剂紫杉醇保存MT，Rho抑制(通过C3外毒素或显性负性(DN)-Rho或dN-Rho-Kinase)减弱ND诱导的TER减少，应激纤维形成和MLC磷酸化。总之，我们的结果表明Rho依赖的机制在MT和肌动蛋白细胞骨架之间的串扰中起主导作用，并提示Rho-K和MYPT1是介导ND诱导的MT解体反应中肺内皮细胞屏障破坏的主要Rho效应因子。
Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.