Wójciak-Stothard B, Entwistle A, Garg R, Ridley A J
Ludwig Institute for Cancer Research, London, United Kingdom.
J Cell Physiol. 1998 Jul;176(1):150-65. doi: 10.1002/(SICI)1097-4652(199807)176:1<150::AID-JCP17>3.0.CO;2-B.
We have investigated the role of the small guanosine-trisphosphate (GTP)-binding proteins, Rho, Rac, and Cdc42, in the early responses of human umbilical vein endothelial cells (HUVECs) to TNF-alpha (tumor necrosis factor-alpha). Quiescent confluent HUVECs incubated with TNF-alpha for 5-30 min showed an increased formation of membrane ruffles, filopodia, and actin stress fibres followed by cell retraction and formation of intercellular gaps. This process was accompanied by the dispersion of cadherin-5 from intercellular junctions. TNF-alpha also induced a transient increase in polymerized F-actin, as determined both by measuring G-actin content and by quantifying fluorescent emission from fluorescein isothiocyanate (FITC)-phalloidin-labelled F-actin. Microinjection of cells with activated RhoA protein led to an increase in polymerized actin, formation of stress fibres, cell retraction as well as dispersion of cadherin-5. The proteins Cdc42 and Rac induced qualitatively similar effects to Rho, although not as dramatic and in addition induced formation of filopodia and lamellipodia. Microinjection of cells with a Rho inhibitor, C3 transferase, prevented gap formation caused by TNF-alpha. Similar effects were observed in cells microinjected with the dominant inhibitory proteins N17Cdc42 and N17Rac1. Cell retraction and gap formation were also prevented by inhibitors of myosin light chain kinase (MLCK). Our data suggest that Cdc42, Rac, and Rho are activated in a hierarchical cascade following stimulation with TNF-alpha leading to actomyosin-mediated cell retraction and formation of intercellular gaps.
我们研究了小GTP结合蛋白Rho、Rac和Cdc42在人脐静脉内皮细胞（HUVECs）对肿瘤坏死因子-α（TNF-α）早期反应中的作用。用TNF-α孵育5-30分钟的静止汇合HUVECs显示，膜皱褶、丝状伪足和肌动蛋白应力纤维形成增加，随后细胞收缩并形成细胞间隙。这一过程伴随着钙黏蛋白-5从细胞间连接处的分散。TNF-α还诱导了聚合F-肌动蛋白的短暂增加，这通过测量G-肌动蛋白含量以及量化异硫氰酸荧光素（FITC）-鬼笔环肽标记的F-肌动蛋白的荧光发射来确定。用活化的RhoA蛋白显微注射细胞导致聚合肌动蛋白增加、应力纤维形成、细胞收缩以及钙黏蛋白-5的分散。Cdc42和Rac蛋白诱导的效应与Rho定性相似，尽管不那么显著，此外还诱导了丝状伪足和片状伪足的形成。用Rho抑制剂C3转移酶显微注射细胞可防止TNF-α引起的间隙形成。在用显性抑制蛋白N17Cdc42和N17Rac1显微注射的细胞中也观察到了类似的效应。肌球蛋白轻链激酶（MLCK）抑制剂也可防止细胞收缩和间隙形成。我们的数据表明，在用TNF-α刺激后，Cdc42、Rac和Rho在一个分级级联中被激活，导致肌动球蛋白介导的细胞收缩和细胞间隙形成。
