Requirement for RhoA kinase activation in leukocyte de-adhesion.
Leukocyte migration from bloodstream to tissue requires rapid coordinated regulation of integrin-dependent adhesion and de-adhesion. Whether de-adhesion is an active process mediated by a distinct signaling pathway(s) or a passive decay of initial adhesion remains undetermined. We found that blockade of RhoA with C3 exoenzyme or inhibition of RhoA kinase by the specific inhibitor Y-27632 enhanced phorbol ester-stimulated alpha(4)beta(1)-dependent adhesion of Jurkat cells at 30 min. Similarly, Y-27632 treatment increased stimulated beta(2) integrin-dependent neutrophil adhesion at 30 min but not at 5 min. Because reduced de-adhesion could mimic augmentation of adhesion at later time points, we developed an assay to measure de-adhesion specifically. Treatment of phorbol ester-or bacterial chemoattractant peptide-but not Mn(2+)-stimulated neutrophils adherent to serum-coated plastic or endothelial cells with Y-27632 or C3 exoenzyme markedly reduced the rate of de-adhesion, while markedly increasing their spreading. RhoA kinase inhibitor effects on de-adhesion and spreading were reversed by treatment with the cytoskeletal-disrupting agent cytochalasin D. Treatment with Y-27632 influenced neither integrin activation epitope nor integrin clustering. We conclude that activation of RhoA kinase promotes leukocyte de-adhesion by inhibiting cytoskeletal-dependent spreading, and that these effects of RhoA kinase constitute a new mechanism for regulation of integrin receptor avidity.
Liu
Li
L
Department of Medicine, Division of Hematology, University of Washington, Seattle 98104, USA. liliu@u.washington.edu
Schwartz
Barbara R
BR
Lin
Nancy
N
Winn
Robert K
RK
Harlan
John M
JM
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001905:Botulinum Toxins; D018821:CD18 Antigens / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019012:Integrin beta1 / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D007962:Leukocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
D000577:Amides; D018821:CD18 Antigens; D004791:Enzyme Inhibitors; D019012:Integrin beta1; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate