Rho and Rac but not Cdc42 regulate endothelial cell permeability.
Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the Rho family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of Rho prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a Rho kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that Rho and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.
Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, London W1W 7BS, UK.
B
B
Wójciak-Stothard
S
S
Potempa
T
T
Eichholtz
A J
AJ
Ridley
D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000187:drug effects; D000577:Amides / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D000199:Actins; D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D006632:Histamine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein