PKCδ regulates endothelial basal barrier function through modulation of RhoA GTPase activity

We have shown that PKC delta 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 PKC delta modulation of barrier function in unstimulated endothelial cell monolayers by assessing the effects of PKC delta inhibition in endothelial cells (EC) derived from rat pulmonary artery (PAEC) and epididymus (FPEC). Rottlerin exposure or Ad PKC delta dn infection significantly enhanced monolayer permeability in both EC. Immunofluorescence analyses demonstrated fewer stress fibers and focal contacts in rottlerin-treated or Ad PKC delta dn-infected EC; yet, PKC delta 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 PKC delta dominant negative protein. Additionally, PKC delta immunoprecipitated with p190RhoGAP and p120RasGAP, modulators of RhoA activity. Thus, PKC delta may regulate basal endothelial barrier function by stabilizing microfilaments and focal contacts by regulating RhoA GTPase activity through upstream modulators, p190RhoGAP and p120RasGAP. (c) 2005 Elsevier Inc. All rights reserved.