The Effects Of P120on NF-κB Activation In Inflammatory Responses Of Human Bronchial Epithelial Cells And Its Possible Mechanisms

ObjectiveTo detect the changes of p120-catenin (p120) expression, the activation of nuclear factor-κB (NF-κB) signaling pathway and the changes of cytokine IL-8expression in lipopolysaccharide (LPS)-provoked inflammatory responses in human bronchial epithelial cells (HBECs), to explore the mechanisms of airway inflammation.MethodsAccording to MTT analysis and our previous research,20μg/ml LPS was chosen as stimuli to establish airway inflammation model in HBECs in vitro. In the model of airway inflammation induced by LPS in HBECs, the expression of p120, IκBα and NF-κB p65was examined by fluorescence quantitative-PCR, immunofluorescence and Western blot. Then, NF-κB p65nuclear translocation was detected by isolation of cytoplasmic and nuclear proteins. Next, IL-8expression was examined by fluorescence quantitative real-time-PCR and enzyme-linked immunosorbent assay (ELISA). Finally, transient transfection and small interfering RNA were used for p120over-expression and knock-down to study the effects of p120on NF-κB signaling pathway.Results Ⅰ. Western blot, immunofluorescence and fluorescence quantitative-PCR resultsshowed that p120was expressed in HBECs, and down-regulated after LPS treatment.Ⅱ. Isolation of cytoplasm and nuclear proteins combined with Western blot found that NF-κB signaling was activated in LPS-treated HBECs. After LPS treatment, NF-κB expression was increased accompanied with IκBα degradation and p65nuclear translocation. ELISA and fluorescent quantitative real-time PCR results showed that IL-8, a pro-inflammatory factor, which is one of the NF-κB target gene products, was also significantly increased.Ⅲ. To further confirm the relationship between p120isoforms and NF-κB signaling pathway in bronchial epithelial cells, p120isoform plasmids and p120small interfering RNA were transiently transfected into HBECs. The results showed that both NF-κB and IL-8had no significant change without p65nuclear translocation. But after LPS treatment, IL-8expression of p120-overexpression group was apparently decreased (P<0.001). On the contrary, p120knock-down activated NF-κB signaling pathway, and NF-κB p65level was increased sharply with p65nuclear translocation and significant up-regulation of IL-8.ConclusionIn LPS-induced airway inflammatory responses, p120is down-regulated, and NF-κB signal pathway is activated. p120over-expression may inhibit NF-κB signaling, whereas p120konckdown significantly activate it. These data strongly suggest that LPS-provoked airway inflammation may result from p120modulating NF-κB activation. ObjectiveTo investigate the changes of RhoA activity and its relationship with pi20and NF-κB in human bronchial epithelial inflammation induced by LPS or scratching, and to explore the possible molecular mechanisms of p120modulating NF-κB activation in airway inflammation.MethodsCo-immunoprecipitation and Western blot were used to detecte the changes of RhoA expression and its binding with p120after LPS treatment or scratching. Then, G-LISA was used to examine the relative level of active RhoA. Next, by isolation of cytoplasmic and nuclear proteins, the changes of NF-κB activity were detected after RhoA inhibition by ROCK inhibitor Y27632. Finally, transient transfection and small interfering RNA were used for p120over-expression and knock-down to detect the changes of RhoA activity and its relationship with p120and NF-κB in human bronchial epithelial inflammation.ResultsⅠ. Through co-immunoprecipitation, we found for the first time that RhoA could directly co-precipitate with p120in HBECs.Ⅱ. After LPS treatment or scratching, although the total RhoA and p120-binded RhoA were unchanged by Western blot and co-immunoprecipitation, the RhoA activity was increased dramatically by G-LISA analysis.Ⅲ. Y27632, a ROCK inhibitor, could inhibit the RhoA activity and partially inhibit nuclear translocation of p65by using isolation of cytoplasm and nuclear proteins.Ⅳ. Upon p120overexpression, although RhoA activity was unchanged, after LPS treatment, RhoA activity of p120-overexpressed group was significantly decreased (P<0.001), compared with control group. Meanwhile, IL-8expression was down regulated (P<0.05).Ⅴ. After p120knock-down, total RhoA was unchanged, and p120-binded RhoA was undetected with loss of p120. But RhoA activity was significantly elevated by GLISA analysis. The elevation became more apparently after LPS treatment and scratching. Meanwhile, IL-8expression was significantly up-regulated after scratching.ConclusionⅠ.In HBECs, RhoA binds with p120. Although total RhoA and p120-binded RhoA are unchanged, RhoA is activated in airway inflammatory responses.Ⅱ. NF-2κB activation is partially inhibited after RhoA inactivation by Y27632.Ⅲ-P120overexpression may not activate RhoA, but p120konck-down does increase RhoA activity, p65nuclear translocation and IL-8expression.In summary, p120may modulate NF-κB signaling pathway partially through RhoA in airway inflammation.