Guo Fukun, Debidda Marcella, Yang Linda, Williams David A, Zheng Yi
Division of Experimental Hematology, Children's Hospital Research Foundation, University of Cincinnati, Cincinnati, Ohio 45229, USA.
J Biol Chem. 2006 Jul 7;281(27):18652-9. doi: 10.1074/jbc.M603508200. Epub 2006 May 11.
Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.
Rac1是一种调节多种细胞功能的细胞内信号转导分子。以往通过在克隆细胞系中过表达Rac1的显性负性或组成型激活突变体的研究表明，Rac1在肌动蛋白片足诱导、细胞-基质黏附及细胞失巢凋亡中起关键作用。在本研究中，我们检测了经Cre重组酶介导删除Rac1基因后，Rac1基因敲除的原代小鼠胚胎成纤维细胞（MEF）的细胞行为。Rac1基因缺失的MEF形态上变得收缩且拉长，在对血小板衍生生长因子或血清的反应中，其片足形成、细胞铺展、细胞-纤连蛋白黏附及黏着斑形成存在缺陷。出乎意料的是，Rac1的缺失也消除了细胞中的肌动蛋白应力纤维，而内源性RhoA活性未检测到改变。尽管黏着斑复合体组分如Src、FAK和纽蛋白的表达和/或激活状态不受Rac1缺失的影响，但黏着斑的数量和大小显著减少，且在Rac1基因缺失的细胞中，Src、FAK和纽蛋白之间的分子复合体被拆解。活性RhoA突变体或ROK的过表达未能挽救Rac1基因缺失细胞的应力纤维和黏着斑缺陷。尽管在血清刺激下，Rac1缺失导致磷酸化PAK1、-AKT和-ERK显著减少，但活性PAK1的重建能够挽救Rac1缺陷细胞的肌动蛋白细胞骨架和黏附表型，而AKT或MEK1则不能。此外，Rac1缺失导致自发凋亡显著增加，活性PAK1、AKT或MEK1的表达可挽救这一现象。我们从基因敲除的原代MEF获得的结果表明，Rac1不仅对片足诱导至关重要，而且对RhoA调节的肌动蛋白应力纤维和黏着斑复合体形成也至关重要，并且Rac1通过失巢凋亡依赖性及非依赖性机制参与细胞存活调节。
