title: Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP: role in regulation of endothelial permeability.
abstract: The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, The University of Illinois, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Holinstat
authors.forename: Nebojsa
authors.initials: N
authors.lastname: Knezevic
authors.forename: Michael
authors.initials: M
authors.lastname: Broman
authors.forename: Allen M
authors.initials: AM
authors.lastname: Samarel
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Dolly
authors.initials: D
authors.lastname: Mehta
mesh_terms: D000199:Actins / Q000737:chemistry; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002448:Cell Adhesion; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008297:Male; D051379:Mice; D008833:Microcirculation; D010539:Permeability; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D013917:Thrombin / Q000737:chemistry; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism / Q000502:physiology*
chemical_list: C074560:ARHGAP35 protein, human; D000199:Actins; C513041:Arhgap35 protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D013917:Thrombin; D001905:Botulinum Toxins; D020558:GTP Phosphohydrolases; D020742:rhoA GTP-Binding Protein