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.
Subject(s)
Basement Membrane/enzymology , Capillary Permeability/physiology , Endothelial Cells/enzymology , Microcirculation/enzymology , Protein Kinase C/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/metabolism , Animals , Cell Membrane Permeability/physiology , Cells, Cultured , DNA-Binding Proteins , Endothelial Cells/cytology , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Focal Adhesions/metabolism , Gene Expression Regulation/physiology , Guanine Nucleotide Exchange Factors/metabolism , Microcirculation/cytology , Mutation/genetics , Nuclear Proteins/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Kinase C-delta , Protein-Tyrosine Kinases/metabolism , Rats , Repressor Proteins , Stress Fibers/metabolism , Transfection , p120 GTPase Activating Protein/metabolism , rhoA GTP-Binding Protein/geneticsABSTRACT
Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKC alpha (PKC alpha EC), PKC delta (PKC delta EC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKC alpha EC. Conversely, the basal level of permeability was significantly blunted in PKC delta EC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Gö6976 and rottlerin, reversed the effects of PKC alpha and PKC delta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKC alpha EC, an effect blocked by pretreatment with Gö6976. PKC delta EC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKC delta EC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKC delta EC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC alpha promotes endothelial barrier dysfunction and PKC delta enhances basal endothelial barrier function.
