RhoA inactivation enhances endothelial barrier function.
The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA. Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a size-selective permeability assay and quantitative digital imaging measurements of SF/FA. C3 treatment disassembled SF/FA, stimulated diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa proteins that included p125(FAK). C3-treated monolayers displayed a 60-85% decline in F-actin content and a 170-300% increase in EC surface area with enhanced endothelial barrier function. This activity correlated with reorganization of F-actin and PY protein(s) to beta-catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation of myosin ribbons, SF/FA, and the increased PY content of proteins, these characteristics were Rho dependent. Our data show that C3 inhibition of Rho proteins leads to cAMP-like characteristics of reduced SF/FA and enhanced endothelial barrier function.
Carbajal
J M
JM
Department of Physiology, University of Arizona, The Benjamin W. Zweifach Microcirculation Laboratories, Department of Veterans Affairs Medical Center, Tucson, Arizona 85723, USA.
Schaeffer
R C
RC
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000199:Actins / Q000032:analysis / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D001905:Botulinum Toxins; D002417:Cattle; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006490:Hemostatics / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007365:Intercellular Junctions / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000032:analysis / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011651:Pulmonary Artery / Q000166:cytology; D013314:Stress, Mechanical; D013917:Thrombin / Q000494:pharmacology; D015534:Trans-Activators; D014443:Tyrosine / Q000378:metabolism; D051176:beta Catenin; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000199:Actins; D003598:Cytoskeletal Proteins; D006490:Hemostatics; D051419:Paxillin; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D015534:Trans-Activators; D051176:beta Catenin; D000246:Adenosine Diphosphate Ribose; D019000:Phosphotyrosine; D014443:Tyrosine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D013917:Thrombin; D001905:Botulinum Toxins; D009218:Myosins; D020742:rhoA GTP-Binding Protein