The Role Of P120-Catenin In Cigarette Smoke Induced Inflammatory Responses Of Human Bronchial Epithelial Cells And Its Possible Mechanisms

PartⅠ The role of p120activating NF-κB signaling pathway in cigarette smoke induced inflammatory responses of airway epithelial cellsObjectiveThe purpose of this study is to detect the expression changes of p120and its effects on NF-κB signaling pathway in cigarette smoke extract (CSE) induced airway inflammatory responses, to preliminarily explore the molecular mechanisms of airway inflammation caused by cigarette smoke.Methods100%CSE solution was prepared according to our laboratory experience and related documents. Firstly, MTT method was used to detect the effects of different concentrated CSE on cell vitality. According to MTT results,15%CSE was chose to stimulate bronchial epithelial cells for different time, then expression of p120, IκBα and p-lκBα were detected by Western blot and p120mRNA was detected by Real-time PCR. The distribution and expression of NF-κB p65was examined by immunofluorescence and isolation of cytoplasmic and nuclear extracts. Next, IL-8protein and mRNA were detected by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (Real-time PCR). Lastly, p120siRNAor transient transfection plasmids of p1201A and3A to knock down or over-express p120was used to examine the effects of p120changes on NF-κB signaling pathway.Results1. MTT results showed that cell vitality remained80%after15%CSE stimulation for24h, but decreased to40%after20%CSE treatment. Western blot found that p120isoform3was constantly detected in human airway epithelial cells and p120was gradually reduced in a concentration and time dependent manner. Real-Time PCR results showed that p120mRNA was also decreased.2. Western blot found that IκBα was reduced while p-IκBα was increased after15%CSE stimulation. Western blot with cytoplasmic and nuclear extracts revealed that nuclear expression of NF-κB p65was increased, whereas the cytoplasmic was decreased. Immunofluorescence also found NF-κB p65nuclear translocation. At the same time, Real-Time PCR and ELISA indicated that IL-8mRNA and protein were also significantly increased.3. After p120silence by siRNA in airway epithelial cells and stimulated with15%CSE, Western blot results showed that although total NF-κB p65was not changed, the phosphorylation at Ser536was significantly increased by ELISA (P <0.05). ELISA assay found that inflammatory factors IL-8, IL-6and IL-1β were significantly increased compared with Scramble group.(P<0.001).4. After transfected with p120 1A and3A plasmids and treated with15%CSE, Western blot showed that although total NF-κB remain unchanged, NF-κB phosphorylation at Ser536was significantly decreased by ELISA (P<0.05). Inflammatory factors IL-8, IL-6and IL-1β were also significantly decreased after p120over-expression by ELISA compared with MT group (P<0.001).ConclusionsIn airway inflammatory responses induced by CSE, p120may regulate airway inflammatoty responses via NF-κB signaling pathway. Part Ⅱ The possible mechanisms of p120activating NF-κB signaling pathway in airway inflammatory responsesObjectiveThe purpose of this study is to further explore the possible molecular mechanisms of p120activating NF-κB signaling pathway in airway inflammatory responses, by detecting the activity and expression of RhoA/ROCK and the expression of another adhesion molecule E-cadherin after CSE stimulation.MethodsCSE was used to establish airway inflammatory responses in vitro, ROCK inhibitors Y27632was added to inhibit RhoA/ROCK signaling pathway, p120siRNA and p120 3A plasmid were transfected to silence or over-express p120, p120DRDD-GFP (unable to bind RhoA) and p120DEcad-GFP (unable to bind E-cadherin) were also transfected, then the expression changes of p120, E-cadherin, NF-κB p65, RhoA and ROCK1were detected by Western blot, the interaction between p120and RhoA was examined by co-immunoprecipitation, the distribution of NF-κB p65and phosphorylation at Ser536were observed by immunofluorescence and ELISA and RhoA activity was detected by G-LISA.Results1. After15%CSE exposure, Western blot results showed that although total RhoA was not changed, the expression of ROCK1was increased, and E-cadherin expression was reduced.2. Co-immunoprecipitation results showed that p120and RhoA were co-existed in one complex in airway epithelial cells. Although the changes of p120binded RhoA was not obvious, RhoA activity was significantly increased by G-LISA after CSE stimulation.3. After p120silence and CSE treatment, Western blot showed that RhoA remained unchanged, but ROCK1was increased and RhoA activity was significantly increased by G-LISA (P<0.001). After over-expression of p1203A, Western blot showed that both RhoA and ROCK1were reduced and RhoA activity was significantly reduced compared with MT group by G-LISA.4. After RhoA/RCOKl signaling pathway was inhibited by Y27632, immunofluorescence revealed that NF-κB p65nuclear translocation was partially inhibited and ELISA results found that phosphorylation at p65Ser (536) was reduced.5. After p120DRDD-GFP transfection, Western blot showed that both RhoA and ROCK1expression were not changed even stimulated with CSE. The phosphorylation of NF-κB p65at Ser (536) and RhoA activity were also not changed. While after p120DEcad-GFP transfection and CSE treatment, Western blot showed that NF-κB remained unchanged, but RhoA was decreased, and the phosphorylation of NF-κB p65at Ser (536) and RhoA activity were both decreased.Conclusions1. p120can bind with RhoA in human airway epithelial cells and RhoA/ROCK signal was activated after CSE stimulation.2. p120silence can enhance the activation of Rho A/ROCK signaling pathway, whereas over-expression of p1203A can partially inhibit the CSE induced activation of Rho A/ROCK signal.3. After inhibition of RhoA/ROCK signaling pathway by Y27632, CSE induced NF-κB activation was partially inhibited.4. p120can not activate NF-κB signaling pathway when not binding with RhoA, while it can still activate NF-κB signaling pathway without E-cadherin.These results suggest that p120may activate NF-κB via RhoA/ROCK signaling but not E-cadherin in airway inflammatory responses.