RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

• Published in: American Journal of Physiology: Cell Physiology Vol. 287; no. 2; pp. C327 - C335
• Main Authors: Bruewer, Matthias, Hopkins, Ann M, Hobert, Michael E, Nusrat, Asma, Madara, James L
• Format: Journal Article
• Language: English
• Published: United States 01.08.2004
• Subjects: Actins - metabolism; Adherens Junctions - metabolism; Animals; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Cell Adhesion Molecules - metabolism; Cell Membrane Permeability - physiology; Claudin-1; Claudins; Detergents; Dogs; Epithelial Cells - metabolism; Kidney - cytology; Membrane Microdomains - metabolism; Membrane Proteins - metabolism; Mutagenesis - physiology; Phosphoproteins - metabolism; rac1 GTP-Binding Protein - genetics; rac1 GTP-Binding Protein - metabolism; Receptors, Cell Surface - metabolism; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; Solubility; Tight Junctions - metabolism; Zonula Occludens-1 Protein
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00087.2004

1 Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322; 2 Department of General Surgery, University of Muenster, 48149 Muenster, Germany; and 3 Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637 Submitted 11 February 2004 ; accepted in final form 22 March 2004 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. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells Address for reprint requests and other correspondence: M. Bruewer, Dept. of General Surgery, Univ. of Muenster, Waldeyerstr. 1, 48149 Muenster, Germany (E-mail: bruwer{at}uni-muenster.de ).