The role of VASP in regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization.
Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP(-/-)) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP(-/-) and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In VASP(-/-) cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased approximately 20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.
Schlegel
N
N
Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany
Burger
S
S
Golenhofen
N
N
Walter
U
U
Drenckhahn
D
D
Waschke
J
J
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001427:Bacterial Toxins / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003331:Coronary Vessels / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051356:Cortactin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000494:pharmacology; D007365:Intercellular Junctions / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D000098662:Vasodilator-Stimulated Phosphoprotein; D000099152:Cadherin 5
D000262:Adenylyl Cyclases; D015703:Antigens, CD; D001427:Bacterial Toxins; D015820:Cadherins; D015815:Cell Adhesion Molecules; D005576:Colforsin; D051356:Cortactin; D000242:Cyclic AMP; D020536:Enzyme Activators; D029968:Escherichia coli Proteins; D008840:Microfilament Proteins; D009479:Neuropeptides; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D020889:Rolipram; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000098662:Vasodilator-Stimulated Phosphoprotein; D000099152:Cadherin 5; C495869:Cttn protein, mouse; C484602:Rac1 protein, mouse; C060296:cytotoxic necrotizing factor type 1