RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin
RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin
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DOI：
10.1152/ajpcell.00087.2004
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发表时间：
2004-08-01
影响因子：
5.5
通讯作者：
Madara, JL
中科院分区：
文献类型：
--
作者：
Bruewer, M;Hopkins, AM;Madara, JL
上皮细胞间连接调节细胞与细胞之间的接触和粘膜屏障功能。紧密连接(TJ)和黏附连接(AJ)都部分地受它们与F-肌动蛋白细胞骨架的联系的调节。细胞骨架又受到Rho家族小GTP酶的影响，如RhoA、rac1和CDC42，所有这些都构成了几种病原体的真核靶标。利用四环素可阻抑系统在马丁达比犬肾(MDCK)上皮细胞中实现调控表达，我们使用显性负性(DN)和结构性活性(CA)形式的RhoA、rac1和CDC42作为工具来评估每个GTP酶对上皮结构和屏障功能的精确贡献。所有突变的GTP酶均可引起上皮门功能的时间依赖性破坏，以及顶端和基础F-肌动蛋白池明显的形态改变。在CA RhoA或CA Cdc42存在下，TJ蛋白occludin、ZO-1、claudin-1、claudin-2和JAM-1显著地重新分布，而CA rac1只引起claudins-1和-2的重新分布。除JAM-1外，DNrac1的表达还可诱导Claudins-1和-2的选择性再分布，而DNcc42仅影响Claudin-2，而DNRhoA没有影响。AJ蛋白的定位不受任何突变型GTP酶的影响，但DNrac1诱导E-钙粘素洗涤剂溶解度降低。所有CA GTP酶都增加了Claudins-1和Claudins-2的洗涤剂溶解度，但CA RhoA单独作用减少了Claudin-2和ZO-1对洗涤剂不溶的膜筏的分配。我们得出结论，Rho家族GTP酶通过不同的形态和生化机制调节上皮细胞间连接，屏障功能的紊乱反映了活性/静息GTP酶水平的任何失衡，而不仅仅是GTP酶活性的丧失或增加。
Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active ( CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity.