The Role Of P115RhoGEF/RhoA Pathway In LPS And TNF-α-induced Brain Microvascular Endothelial Barrier Dysfunction

Infectious brain injury is one of the commonest causes of mortality in clinical bacterial infection, despite the availability of effective antibiotic treatment. The reasons for the unfavorable clinical outcome include cerebrovascular alterations, disruption of the blood brain barrier (BBB), and increase in intracranial pressure (ICP). The BBB plays an important role in controlling the access of inflammatory cells and macromolecules into the brain by virtue of tight junctions (TJ) between brain microvascular endothelial cells (BMECs). Several tight junction-associated protein components were identified in cerebral endothelial cell tight junctions, such as occludin, claudins (claudin-1, claudin-5), and zonula occludens (ZO-1, ZO-2, ZO-3). Cytokines such as tumor necrosis factor-a (TNF-a) is released in large amount by leucocytes in response to gram-negative bacterial substances like LPS. There is evidence that LPS and TNF-a involve in alteration of endothelial permeability during infection.RhoA is a prototypical member of the Rho subfamily of Ras-related small GTP-binding proteins and is linked to multiple signal transduction pathways, including cytoskeleton-related events and gene transcription. Recently, the RhoA and Racl small GTPases were shown to play a role in the regulation of tight junction structure and function. Meanwhile, our studies supported that RhoA acts as an important regulator of LPS and TNF-a signaling in endothelia. For example, Rho kinase inhibition (Y-27632) significantly attenuated LPS-induced EC barrier disruption. GEFs (guanine nucleotide exchange factors,) activate RhoA by stimulating release of GDP and allowing GTP to bind RhoA. p115RhoGEF, a specific GEF activates RhoA, triggering RhoA-dependent cytoskeletal remodeling. Although studies in peripheral endothelial cell lines have shown that RhoA is involved in LPS-induced barrier injury and p115RhoGEF is a critical regulator of RhoA, the role of this signaling pathway has not been studied in brain. Therefore, analysis of p115RhoGEF and RhoA regulation initiated by LPS or TNF-α in BBB could lead to an understanding of pathology and to the identification of novel therapeutic targets.Section one The effect of LPS on the barrier function and its mechanisms in rat brain microvascular endothelial cellsObjective:To investigate the effect of LPS on the barrier function, RhoA activity, expression of p115RhoGEF and TJ proteins in rat brain microvascular endothelial cells (BMECs).Methods:Monolayers of primary rat BMECs were cultured and divided randomly into two groups:control group and LPS group. The barrier permeability was measured by transendothelial electrical resistance (TEER) assay. The levels of RhoA activity after LPS (5ug/ml) treatment were determined by Pull-down assay at various time points. The expression of p115RhoGEF, ZO-1, occludin and claudin-5were analyzed by Western blot analysis.Results:The average TEER value of rat BMECs in control group was (159.0±8.6) Ω·cm2, while it decreased apparently to (108.3±4.2)Ω·cm2and (85.4±2.5)Ω·cm2after the cells had been incubated with LPS for3and12hrs. Pull down assessment of RhoA activity showed that LPS activated RhoA rapidly within5minute. p115RhoGEF expression was significantly elevated in BMECs after1hr of treatment with LPS. The protein levels of ZO-1, occludin and claudin-5decreased significantly after LPS treatment (P<0.05).Conclusions:LPS decreases expression of ZO-1, occludin and claudin-5, resulting in an increased permeability of rat BMECs. Section2Role of p115RhoGEF in LPS-induced endothelial barrier dysfunction in Bend.3cellsObjective:To detect the effect of inhibition of p115RhoGEF on the barrier function, activity of RhoA and the expression of ZO-1, occludin and claudin-5in LPS stimulated bEnd.3cell line.Methods:The cultured monolayer of bEnd.3cells, an immortalized mouse brain endothelial cell line, was used cultured. bEnd.3cells were divided randomly into three groups:control group, C3transferase group, p115RhoGEF siRNA group. bEnd.3cells in C3transferase group were pretreated with C3transferase to inhibit RhoA activity, and specific siRNA to knockdown p115RhoGEF was transfected into cells in p115RhoGEF siRNA group by Lipofectamine2000. The activity of RhoA after LPS (5μg/ml) treatment was determined by pull-down assay, and expression of p115RhoGEF, ZO-1, occludin and claudin-5proteins were detected by Western blot analysis. The barrier function was measured by transendothelial electrical resistance (TEER). F-actin cytoskeleton was visualized by Rhodamine-phalloidin staining.Results:The upregulation of p115RhoGEF was detected as early as1h after start of LPS treatment and expression continuously increased up to6h. The depletion of p115RhoGEF protein by siRNA was confirmed by Western blot analysis. RhoA activity was increased115%for5min following treatment with LPS. p115RhoGEF siRNA and C3transferase, respectively, reduced RhoA activation by70%and91%after5min LPS treatment. RhoA activity displayed a remarkable reduction in C3group as compared with siRNA group. Pretreatment of cells with nsRNA alone had no significant effect on TEER. LPS resulted in a time-dependent decrease of TEER in the control group. Pretreatment with p115RhoGEF siRNA and C3transferase significantly abrogated the effect of LPS. TEER of siRNA group and C3group, respectively, were65.4±2.01Ω·cm2and72.3±1.63Ω·cm2compared to55.3±2.08Ω·cm2for nsRNA group at3hr.3-hour LPS exposure caused stress fiber formation and paracellular gaps between cell monolayer, whereas F-actin structure remained unaffected in cells transfected with p115RhoGEF-siRNA and C3transferase. The cellular protein levels of ZO-1, occludin, and claudin-5decreased in a time-dependent manner under treatment with LPS. Loss of ZO-1can be reversed by inhibition of p115RhoGEF. The expression of occludin was a little higher in the siRNA group than in the nsRNA group at6hr and12hr, but there was no statistical difference comparing to nsRNA group. Cellular protein levels of claudin-5were decreased with incubation of LPS for12hr, and p115RhoGEF siRNA failed to reverse the loss of claudin-5proteins compared with control nsRNA group. In contrast, degradation of these three TJ proteins was blocked by C3transferase comparing with nsRNA group and siRNA group (P<0.05). Conclusions:These results suggest that p115RhoGEF is important for LPS signaling to RhoA and LPS-induced endothelial barrier dysfunction, providing new insight into the function of RhoGEFs in inflammation. Section3Rho pathway was involved in TNF-α-induced BMECs hyperpermeabilityOBJECTIVES:To investigate the effects of TNF-α on RhoA activity in mouse cerebral microvascular endothelial cells and to explore the potential mechanisms involved.METHODS:bEnd.3cells, a mouse brain endothelial line, were cultured. RhoA activity was analyzed by pull-down assay at10,30, and60min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting at1,3,6,12, and24h after TNF-a treatment. p115RhoGEF small interfering RNA (siRNA) or control nsRNA was transfected into bEnd.3cells. Then, p115RhoGEF expression was determined using Western blotting and RhoA activity was detected by Pull-down assay at30min after TNF-α treatment. bEnd.3cells were divided randomly into normal control group, TNF-α group and Y-26732pretreatment group. The changes of monolayer permeability were evaluated by TEER.RESULTS:RhoA activity peaked at30min after TNF-α treatment. TNF-α significantly increased the expression of RhoA at12and24h. Inhibition of p115RhoGEF by siRNA in bEnd.3cells attenuated TNF-α- induced RhoA activation. TEER was significantly decreased after TNF-a treatment compared with control group, Y-27632pretreatment could prevent the hyperpermeability of BMECs induced by TNF-α.CONCLUSION TNF-α can up-regulate RhoA activity and expression. p115RhoGEF/RhoA signaling pathway is involved in the regulation of bEnd.3permeability induced by TNF-α.