RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability.
Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.
Clements
Richard T
RT
Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA.
Minnear
Fred L
FL
Singer
Harold A
HA
Keller
Rebecca S
RS
Vincent
Peter A
PA
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011651:Pulmonary Artery / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology
D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011725:Pyridines; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C108830:Y 27632; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein