Birukov Konstantin G, Birukova Anna A, Dudek Steven M, Verin Alexander D, Crow Michael T, Zhan Xi, DePaola Natacha, Garcia Joe G N
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Am J Respir Cell Mol Biol. 2002 Apr;26(4):453-64. doi: 10.1165/ajrcmb.26.4.4725.
Hemodynamic forces in the form of shear stress (SS) and mechanical strain imposed by circulating blood are recognized factors involved in the control of systemic endothelial cell (EC) cytoskeletal structure and function. However, the effects of acute SS on pulmonary endothelium have not been precisely characterized, nor the mechanism of rapid SS-induced EC cytoskeletal rearrangement understood. We exposed bovine and human pulmonary EC monolayers to laminar SS (10 dynes/cm2) in a parallel plate flow chamber and observed increased actin stress fiber formation 15 min after application of flow. Acute SS-induced pronounced cortical cytoskeletal rearrangement characterized by myosin light chain kinase (MLCK)- and Rho-associated kinase (RhoK)-dependent accumulation of diphosphorylated regulatory myosin light chains (MLC) in the cortical actin ring, junctional protein tyrosine phosphorylation, and transient peripheral translocation of cortactin, an actin-binding protein involved in the regulation of actin polymerization. SS-induced cortactin translocation was independent of Erk-1,2 MAP kinase, p60(Src), MLCK, or RhoK activities, and unaffected by overexpression of a cortactin mutant lacking four major p60(Src) phosphorylation sites. However, both SS-induced transient cortactin translocation and cytoskeletal reorientation in response to sustained (24 h) SS was abolished in cells overexpressing either dominant negative Rac 1 or a dominant negative construct of its downstream target, p21-activated kinase (PAK)-1. Our results suggest a potential role for cortactin in the SS-induced EC cortical cytoskeletal remodeling and demonstrate a novel mechanism of Rac GTPase-dependent regulation of the pulmonary endothelial cytoskeleton by SS.
以剪切应力（SS）形式存在的血流动力学力以及循环血液施加的机械应变是参与调控全身内皮细胞（EC）细胞骨架结构和功能的公认因素。然而，急性SS对肺内皮的影响尚未得到精确表征，快速SS诱导的EC细胞骨架重排机制也未被理解。我们将牛和人肺EC单层细胞置于平行板流动腔中，使其暴露于层流SS（10达因/平方厘米），并观察到施加流动15分钟后肌动蛋白应力纤维形成增加。急性SS诱导明显的皮质细胞骨架重排，其特征为在皮质肌动蛋白环中，依赖肌球蛋白轻链激酶（MLCK）和Rho相关激酶（RhoK）的双磷酸化调节性肌球蛋白轻链（MLC）积累、连接蛋白酪氨酸磷酸化以及肌动蛋白结合蛋白cortactin（参与肌动蛋白聚合调节）的短暂外周易位。SS诱导的cortactin易位独立于Erk-1,2丝裂原活化蛋白激酶、p60(Src)、MLCK或RhoK活性，并且不受缺乏四个主要p60(Src)磷酸化位点的cortactin突变体过表达的影响。然而，在过表达显性负性Rac 1或其下游靶点p21激活激酶（PAK）-1的显性负性构建体的细胞中，SS诱导的短暂cortactin易位和对持续（24小时）SS的细胞骨架重新定向均被消除。我们的结果表明cortactin在SS诱导的EC皮质细胞骨架重塑中具有潜在作用，并证明了一种SS依赖的Rac GTP酶调控肺内皮细胞骨架的新机制。
