Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

Abstract

BackgroundTumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC) isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC) barrier dysfunction.MethodsBend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL). RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER). p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability.ResultsWe observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA) transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER.ConclusionsTaken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF-α signaling to RhoA, and that this plays a critical role in signaling F-actin rearrangement and barrier dysfunction in BMECs.

Interleukin-1β-induced barrier dysfunction is signaled through PKC-θ in human brain microvascular endothelium

“…Likewise, the isoform-selective PKC inhibitor Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] prevented microvascular leakage during ischemia-reperfusion injury in the rat heart, as well as hyperpermeability in human coronary microvascular endothelium in response to IL-1␤ exposure (36). In addition, BBB dysfunction was mediated by PKC-␣ in response to tumor necrosis factor (TNF)-␣ exposure in mouse brain endothelium (29) and involved activation of PKC-and possibly other PKC isoforms during hypoxia in the rat brain (18,37). Based on these observations, we hypothesized that select PKC isoforms mediate hyperpermeability in human brain microvascular endothelium during IL-1␤ exposure.…”

Reactive Oxygen Species in Inflammation and Tissue Injury

“…These modifications further increase kinase activity of PKC (243). Several PKC inhibitors such as staurosporine, H7, calphostin C, bisindolylmaleimide, and chelerytherine inhibited endothelial hyper-permeability in response to different pro-inflammatory agents (133,200,341,358,460). A number of studies have demonstrated that membrane translocation and activation of the atypical isoforms of PKCf is required in thrombin-mediated increase in endothelial permeability (244,294).…”

Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects

“…More recently, Arghef1 was also identified as the RhoA GEF mediated-RhoA activation and permeability induced by TNF-␣ (361) and LPS (546). Activation of Arhgef1 by TNF-␣ is mediated by PKC␣ phosphorylation of Arhgef1 (361).…”