Clements Richard T, Minnear Fred L, Singer Harold A, Keller Rebecca S, Vincent Peter A
Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA.
Am J Physiol Lung Cell Mol Physiol. 2005 Feb;288(2):L294-306. doi: 10.1152/ajplung.00213.2004. Epub 2004 Oct 8.
Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.
转化生长因子（TGF）-β是一种参与急性肺损伤的强效炎症介质。TGF-β直接增加肺内皮肌球蛋白轻链（MLC）磷酸化，这与内皮应力纤维形成增加、间隙形成和蛋白质通透性增加有关，这些都是急性肺损伤期间肺内皮反应的特征。我们在肺内皮单层中进行了以下实验，以确定RhoA和Rho激酶是否介导这些TGF-β诱导的反应。TGF-β在处理后2小时导致RhoA持续激活，同时MLC磷酸化增加。用C3外切酶或Y-27632抑制RhoA或Rho激酶均可阻断MLC磷酸化。此外，C3和Y-27632均部分减弱了TGF-β诱导的通透性最大增加，但不影响屏障完整性受损的初始阶段。抑制Rho激酶完全阻断了TGF-β诱导的丝状肌动蛋白（F-肌动蛋白）含量增加，但仅部分抑制了TGF-β诱导的肌动蛋白重组变化。为了评估Rho激酶在独立于其他TGF-β诱导信号的RhoA介导反应中的作用，细胞用组成型活性RhoA腺病毒（RhoAQ63L）感染，同时或不同时使用Y-27632。RhoAQ63L增加了MLC磷酸化、F-肌动蛋白含量和通透性。用Y-27632处理可阻断这些反应，表明Rho激酶介导这些RhoA诱导的效应。总体而言，这些数据表明：1）RhoA/Rho激酶途径是TGF-β诱导的对内皮MLC磷酸化、细胞骨架重组和屏障完整性影响的重要组成部分；2）独立于RhoA/Rho激酶信号级联的其他信号机制促成了TGF-β诱导的细胞骨架组织和通透性变化。
