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

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