Role of p115RhoGEF in lipopolysaccharide-induced mouse brain microvascular endothelial barrier dysfunction

BackgroundIn endothelial cells, exposure to lipopolysaccharide (LPS) results in barrier dysfunction through a complex signaling mechanism. The RhoA/Rho-kinase pathway plays a significant role in endothelial cell permeability. p115RhoGEF, a specific guanine nucleotide exchange factors (GEFs) activates RhoA, triggering RhoA-dependent cytoskeletal remodeling. However, little is known about the role of p115RhoGEF in LPS-induced brain endothelial barrier breakdown. We hypothesized that suppression of p115RhoGEF may inhibit activation of RhoA and prevent LPS-induced brain microvascular endothelial cell hyperpermeability.MethodsThe cultured monolayer of bEnd.3 cells, an immortalized mouse brain endothelial cell line, was used in this study. bEnd.3 cells were pretreated with specific siRNA to knockdown p115RhoGEF or C3 transferase to inhibit RhoA activity, and then incubated with LPS (5 μg/ml). The degree of RhoA activation was determined by a Rhotekin-based pull-down assay, and expression of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins 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.ResultsThe expression level of p115RhoGEF protein was significantly increased in LPS-treated bEnd.3 cells. The activity of RhoA was enhanced after LPS stimulation and pretreatment with p115RhoGEF siRNA or exoenzyme C3 transferase reduced RhoA activation significantly as shown by the pull-down assay. Furthermore, depletion of p115RhoGEF partially prevented the LPS-induced decrease in TEER, stress fiber formation and tight junction proteins degradation.ConclusionsThese 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.

► LPS up-regulates the expression of p115RhoGEF.
► p115RhoGEF involved in signaling of LPS regulating RhoA activation in bEnd.3 cells.
► p115RhoGEF plays an important role in signaling increased paracellular permeability.