title: PKCdelta regulates endothelial basal barrier function through modulation of RhoA GTPase activity.
abstract: We have shown that PKCdelta enhanced microvascular endothelial basal barrier function, correlating with elevated RhoA GTPase activity and increased focal contact formation. In the current study, we investigated signaling pathways important in PKCdelta modulation of barrier function in unstimulated endothelial cell monolayers by assessing the effects of PKCdelta inhibition in endothelial cells (EC) derived from rat pulmonary artery (PAEC) and epididymus (FPEC). Rottlerin exposure or Ad PKCdeltadn infection significantly enhanced monolayer permeability in both EC. Immunofluorescence analyses demonstrated fewer stress fibers and focal contacts in rottlerin-treated or Ad PKCdeltadn-infected EC; yet, PKCdelta inhibition caused no significant changes in microtubule structures. These changes correlated with a reduction in both focal adhesion kinase (FAK) and RhoA GTPase activities. Microfilament stabilization significantly attenuated the focal contact and barrier disruptive effects of rottlerin. FAK overexpression did not blunt the effects of rottlerin-induced barrier dysfunction or stress fiber and focal contact disruption. Conversely, GFP-linked dominant active RhoA overexpression protected EC from stress fiber and focal contact disruption induced by both rottlerin exposure and overexpression of PKCdelta dominant negative protein. Additionally, PKCdelta immunoprecipitated with p190RhoGAP and p120RasGAP, modulators of RhoA activity. Thus, PKCdelta may regulate basal endothelial barrier function by stabilizing microfilaments and focal contacts by regulating RhoA GTPase activity through upstream modulators, p190RhoGAP and p120RasGAP.
authors.affiliation: Pulmonary Vascular Research Laboratory, Providence VA Medical Center, Department of Medicine, Brown Medical School, 151 Research Services, 830 Chalkstone Avenue, Providence, RI 02908, USA. Elizabeth_Harrington@brown.edu
authors.forename: Elizabeth O
authors.initials: EO
authors.lastname: Harrington
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Shannon
authors.forename: Nicole
authors.initials: N
authors.lastname: Morin
authors.forename: Heather
authors.initials: H
authors.lastname: Rowlett
authors.forename: Christopher
authors.initials: C
authors.lastname: Murphy
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane / Q000201:enzymology*; D002199:Capillary Permeability / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D008833:Microcirculation / Q000166:cytology / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins; D022502:Stress Fibers / Q000378:metabolism; D014162:Transfection; D020729:p120 GTPase Activating Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C513040:Arhgap35 protein, rat; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D012097:Repressor Proteins; D020729:p120 GTPase Activating Protein; C497260:Prkcd protein, rat; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D020742:rhoA GTP-Binding Protein