Protein Kinase C-α/P115RhoGEF/RhoA Signals TNF-α-Induced Mouse Brain Microvascular Endothelial Cell Barrier Dysfunction

The BMECs, which are linked by tight junctions, act as a physical and metabolic barrier to shield the brain from toxic substances in the blood, supply brain tissues with nutrients, and filter harmful compounds from the brain back to the bloodstream.Tumor necrosis factor-a (TNF-a) is released in large amounts by macrophages, monocytes and other leukocytes in response to gram-positive or gram-negative bacterial substances, and plays a vital role in the pathogenesis of infectious brain edema. We previously demonstrated that elevated TNF-a is highly correlated with the occurrence of blood brain barrier (BBB) dysfunction, and that inhibiting Rho kinase by pretreatment with Y-27632alleviates brain edema in animals after TNF-a challenge. Thus, these findings suggest an essential role for the RhoA/Rho kinase pathway in the regulation of TNF-a-induced BBB dysfunction. However, little is known about the complex signaling events regulating RhoA, by which TNF-a plays a role in BBB dysfunction.Therefore, identification of the inflammatory signaling initiated by TNF-α in BBB is paramount to understanding the mechanisms of infectious brain edema. Section one TNF-α activate RhoA, mediating barrier dysfunction in Bend.3cells.Objective To address the direct involvement of RhoA in TNF-α-induced Bend.3cell barrier dysfunction. Methods PcDNA3.1hygro-n19RhoA(the dominant negative mutant of RhoA), and PcDNA3.1hygro vector plasmids (vector-1) were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The remarkable inhibitory effect of n19RhoA was confirmed by pull-down assay. The level of active RhoA in vector-1cells after TNF-α (10ng/ml) treatment were detected at various time points by pull-down assay to detect relative amounts of RhoA-GTP. F-actin remodeling and paracellular gap formation were detected by Rhodamine-phalloidin staining. Vector-1and n19RhoA transfected cells were treated with or without TNF-α for varying time periods. TER assay was used to determine Bend.3monolayer permeability. Results Pull Down assessment of RhoA activity showed that in the Bend.3/n19rhoa cells, the active GTP-loaded RhoA were much more less than these in the general Bend.3cells and the Bend.3/vector cells. Consquently, the results showed that TNF-α induced a rapid activation of RhoA within1min, reaching peak activation within30min. TNF-α exposure induced changes in F-actin organization, which also appeared in a time-dependent manner. However, TNF-α-induced vector-1cell barrier breakdown was partially alleviated in n19RhoA cells. Conclusions These data indicate that TNF-α activate RhoA, which mediates F-actin remodeling and barrier dysfunction in Bend.3cells. Section two The mechanism of RhoA activation and BMECs barrier dysfunction induced by tumor necrosis factor-aObjective An attempt was made to explore the complex signaling events regulating RhoA, by which TNF-α plays a role in BMEC barrier dysfunction. Methods The Bend.3cells transfected with vector-2, PKC-a-shRNA, PKC-β-shRNA and p115-shRNA respectively. Stably transfected cells were used for experiments after selection by Puromycin (300ug/ml). The inhibitory levels of p115RhoGEF, PKC-a and PKC-β expression were confirmed by Western blot. Bend.3cells were pretreated with Go6976(1μM), a selective inhibitor of conventional PKC isozymes,or transfected with vector-2, PKC-a-shRNA, PKC-β-shRNA and p115-shRNA respectively, then p115RhoGEF phosphorylation and RhoA activation were assayed after TNF-a challenge. Futhermore, serum-starved cells were transfected with vector-1, n19RhoA, vector-2and P115-shRNA. Cells were stimulated with TNF-a for1min, and then PKC-a activation was detected by vitro enzyme assasy. Serum-starved cells were transfected with vector-1, n19RhoA, vector-2, P115-shRNA and PKCa-shRNA. We analyzed the effect of RhoA inactivation, p115RhoGEF and PKC-a knockdown on TNF-a-induced F-actin dynamics by immunofluorescence and barrier permeability by TER. Results Western blot confirmed that the levels of PKC-a, PKC-p and p115RhoGEF expression significantly decreased in PKC-a-shRNA, PKC-β-shRNA or p115-shRNA cells than vector-2cells.We observed that TNF-α induced a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Go6976mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events were regulated by PKC-α rather than PKC-β by using shRNA. In addition, p115-shRNA and n19RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. Furthermore, TNF-α caused a obviously decrease in TER in BMECs transfected with vector-2alone. However, this response was significantly reduced in cells transfected with n19RhoA, p115-shRNA or PKC-α-shRNA. These effects were accompanied by decreases in the amount of stress fibers and paracellular gaps. Conclusions These data suggest that PKC-a but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. PKC-α/p115RhoGEF/RhoA pathway is the mechanism mediating TNF-α-induced dynamics of F-actin and elevation of BMECs permeability, which in turn might contribute to infectious brain edema.Section three PKC-α, p115RhoGEF and RhoA were involved in pathogenesis of blood brain barrier hyperpermeability in E.Coli meningitis rat model Objective To address the direct involvement of PKC-a, p115RhoGEF and RhoA in pathogenesis of blood brain barrier (BBB) dysfunction in rat bacterial meningitis model. Methods Using intracisternal injection method to construct E.Coli meningitis rat model. All of the rats were identified by bacterial cultures, histopathology and behaviour observation in24h after intracisternal injection. Followed experiments were taken on in2groups:meningitis group (M) and control group (C). Evans blue staining was used to determine BBB permeability. The expression of TNF-a were detected by RT-PCR. The degree of RhoA and PKC-a activation were determined by pull-down assay and enzyme activity assay, and expression of p115RhoGEF were detected by western blot analysis. Results BBB permeability of meningitis group was2folds compared with control group. TNF-a increased in meningitis group rats, which was correspondent with activation of RhoA and PKC-a. In addition, p115RhoGEF was up-regulated in meningitis group. Conclusions These data indicate that PKC-a, p115RhoGEF and RhoA were involved in pathogenesis of BBB hyperpermeability in rat bacterial meningitis model.