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.
Lung edema due to increased vascular permeability is a hallmark of acute lung injury and acute respiratory distress syndrome. Both p38 and RhoA signaling events are involved in transforming growth factor (TGF)-beta1-increased endothelial permeability; however, the mechanism by which these pathways cooperate is not clear. In this study, we hypothesized that TGF-beta1-induced changes in endothelial monolayer permeability and in p38 and RhoA activation are dependent on Smad2 signaling. We assessed the role of Smad2 in p38 activation and the role of p38 in RhoA activation by TGF-beta1. We found that TGF-beta1 caused Smad2 phosphorylation between 0.5 and 1 h of exposure in endothelial cells. Knockdown of Smad2 protein prevented TGF-beta1-induced p38 activation and endothelial barrier dysfunction. Furthermore, TGF-beta1-enhanced RhoA activation was dependent on p38 activation. Inhibition of the RhoA-Rho kinase signaling pathway blunted TGF-beta1-induced adherens junction disruption and focal adhesion complex formation. In addition, depletion of heat shock protein 27, a downstream signaling molecule of p38, did not prevent TGF-beta1-induced endothelial barrier dysfunction. Finally, inhibition of de novo protein expression blunted TGF-beta1-induced RhoA activation and endothelial barrier dysfunction. Our data indicate that TGF-beta1 induces endothelial barrier dysfunction involving Smad2-dependent p38 activation, resulting in RhoA activation by possible transcriptional regulation.
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