RhoA and NF-κB are involved in lipopolysaccharide-induced brain microvascular cell line hyperpermeability

[Purpose] The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of brain microvascular cell lines induced by lipopolysaccharide (LPS). [Methods] To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3 cells were exposed to LPS, and changes in endothelial permeability (transendothelial electrical resistance (TEER) assay), F-actin dynamics (Rhodamine-Phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. Moreover, to ensure that both RhoA and NF-κB participated in the regulatory mechanisms, Bend.3 cells were transfected with n19RhoA and DNMu-IκBα plasmids, and the above experiments were repeated. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in transfected and untreated Bend.3 cells. Lastly, to investigate whether RhoA and NF-κB regulate MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation in untreated and transfected Bend.3 cells by western blot. [Result] LPS caused RhoA and NF-κB activation, MLC phosphorylation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were suppressed by inhibitors of RhoA or NF-κB; inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-κB activation, but the inverse was not true. [Conclusions] LPS induces brain microvascular endothelial barrier hyperpermeability by disrupting TJs, in part through RhoA and NF-κB activation, in which RhoA is the positive upstream regulator for NF-κB.

▶Disruption of tight junction relates to LPS-induced Bend.3 cell hyperpermeability. ▶Both RhoA and NF-κB mediates LPS signal to disrupt tight junction in Bend.3 cell. ▶Active RhoA, rather than RhoA silencing, is the upstream signal to activate NF-κB. ▶RhoA/NF-κB/MLC phosphorylation is crucial to regulate tight junction of Bend.3 cell.