title: VEGF activation of protein kinase C stimulates occludin phosphorylation and contributes to endothelial permeability.
abstract: OBJECTIVE
  VEGF is a potent permeabilizing factor that contributes to the pathogenesis of diabetic retinopathy and brain tumors. VEGF-induced vascular permeability in vivo and in cell culture requires PKC activity, but the mechanism by which PKC regulates barrier properties remains unknown. This study was conducted to examine how VEGF and diabetes alter occludin phosphorylation and endothelial cell permeability.
  
  
  METHODS
  Chemical PKC inhibitors and activators were used to treat primary retinal endothelial cells in culture. In vitro kinase assays and Western blot analysis of two-dimensional (2D) and one-dimensional (1D) gel retardation assays were used to analyze occludin phosphorylation. Endothelial cell permeability was determined by measuring the flux of 70-kDa dextran through a cell monolayer in culture. Exogenous expression of a dominant negative PKCbetaII mutant (S217A) was used to assess the PKC dependence of VEGF-induced occludin phosphorylation and endothelial permeability. Occludin phosphorylation was also determined in retinas of streptozotocin-induced diabetic rats.
  
  
  RESULTS
  VEGF stimulated the phosphorylation of occludin in primary retinal endothelial cells. Chemical inhibitors of PKC activity blocked the VEGF-induced increase in occludin phosphorylation, as assessed by 2D gel and gel retardation in Western blot analysis, and blocked part of the VEGF-induced monolayer permeability to 70-kDa dextran. Expression of a dominant negative PKCbetaII mutant blocked VEGF-induced occludin phosphorylation and endothelial permeability. Finally, elevated occludin phosphorylation was observed in the retina of diabetic animals.
  
  
  CONCLUSIONS
  These results strongly suggest that VEGF-induced endothelial permeability requires PKC-dependent phosphorylation of occludin. Regulation of PKC activity and tight junction protein modifications may have therapeutic implications for treatment of diabetic retinopathy and brain tumors.
authors.affiliation: Departments of Cellular and Molecular Physiology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA
authors.forename: Nicole S
authors.initials: NS
authors.lastname: Harhaj
authors.forename: Edward A
authors.initials: EA
authors.lastname: Felinski
authors.forename: Ellen B
authors.initials: EB
authors.lastname: Wolpert
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Sundstrom
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Gardner
authors.forename: David A
authors.initials: DA
authors.lastname: Antonetti
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D003930:Diabetic Retinopathy / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D006854:Hydrocortisone / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568987:Ocln protein, rat; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D011493:Protein Kinase C; D006854:Hydrocortisone