Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent.
Protein kinase C (PKC) is activated in response to various inflammatory mediators and contributes significantly to the endothelial barrier breakdown. However, the mechanisms underlying PKC-mediated permeability regulation are not well understood. We prepared microvascular myocardial endothelial cells from both wild-type (WT) and caveolin-1-deficient mice. Activation of PKC by phorbol myristate acetate (PMA) (100 nM) for 30 min induced intercellular gap formation and fragmentation of VE-cadherin immunoreactivity in WT but not in caveolin-1-deficient monolayers. To test the effect of PKC activation on VE-cadherin-mediated adhesion, we allowed VE-cadherin-coated microbeads to bind to the endothelial cell surface and probed their adhesion by laser tweezers. PMA significantly reduced bead binding to 78+/-6% of controls in WT endothelial cells without any effect in caveolin-1-deficient cells. In WT cells, PMA caused an 86+/-18% increase in FITC-dextran permeability whereas no increase in permeability was observed in caveolin-1-deficient monolayers. Inhibition of PKC by staurosporine (50 nM, 30 min) did not affect barrier functions in both WT and caveolin-1-deficient MyEnd cells. Theses data indicate that PKC activation reduces endothelial barrier functions at least in part by the reduction of VE-cadherin-mediated adhesion and demonstrate that PKC-mediated permeability regulation depends on caveolin-1.
Waschke
Jens
J
Institute of Anatomy and Cell Biology, Julius-Maximilians-University, Koellikerstr. 6, 97070 , Würzburg, Germany. jens.waschke@mail.uni-wuerzburg.de
Golenhofen
Nikola
N
Kurzchalia
Teymuras V
TV
Drenckhahn
Detlev
D
D000818:Animals; D015820:Cadherins / Q000502:physiology; D051242:Caveolin 1 / Q000235:genetics* / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003911:Dextrans; D042783:Endothelial Cells / Q000187:drug effects / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D017629:Gap Junctions / Q000187:drug effects; D015151:Immunoblotting; D007150:Immunohistochemistry; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015820:Cadherins; D051242:Caveolin 1; D003911:Dextrans; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C015219:fluorescein isothiocyanate dextran; D011493:Protein Kinase C; D019311:Staurosporine; D016650:Fluorescein-5-isothiocyanate; D013755:Tetradecanoylphorbol Acetate