Role Of P120-catenin And IQ Domain GTPase-avtivating Protein 1 In The Injury And Repair Of Airway Epithelial Cell

BackgroundAs the first line to defence harmful stimuli, the airway is frequently injured because of its exposure to the external environment. Some pathogenic factors may cause airway inflammation, such as lipopolysaccharide (LPS), which is the main component of the cell wall of Gram-negative bacteria. LPS may promotes several cellular processes including necrosis, apoptosis, and secretion of pro-inflammatory cytokines, which contribute to the development of lung injury. LPS is a potent activator that induces inflammatory gene expression through nuclear factor-KB (NF-κB) activation. NF-κB is a transcription factor expressed ubiquitously, which could be activated by LPS in various cells.NF-κB is normally sequestered in the cytoplasm of resting cells by inhibitor of NF-κB (IκB) and remains transcriptionally inactive. Stimulation by triggers such as LPS induces the ubiquitylation and degradation of IκB. The loss of IκB exposes the nuclear localization signal sequence on NF-κB, resulting in the nuclear translocation of NF-κB and transcriptional activation of its target gene promoters. P120-catenin (p120), a prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins, which is involved in maintaining the stability and regulating the turnover of E-cadherin. Recent studies revealed that loss of p120 was associated with NF-κB activation and inflammation in p120 null epidermal cells. This discovery raised an interest in the pathophysiology of the diseases in which NF-κB activation is involved. Although airway inflammation has been extensively studied, it is uncertain whether p120 partcipates in airway inflammation through NF-κB signaling pathway. Therefore, our present studies focused on the effects of p120 on NF-κB signaling during the lung injury induced by LPS.ObjectiveTo investigate the changes of p120 expression and the effects of p120 on NF-κB signaling pathway during the inflammatory response induced by LPS.MethodsIn this study, we treated cells with LPS to establish a lung inflammation model in vitro. Using confocal immunofluorescence imaging, Western blot, isolation of cytoplasmic and nuclear proteins, we observed and examined the localizations and expressions of p120, NF-κB and IκBα. Then we detected the expressions of IL-8 by fluorescence quantitative PCR and enzyme-linked immunosorbent assay. Luciferase reporter analysis was used to detect the activity of NF-κB. Finally, transient transfection and small interfering RNA were used to over-expression or knock down p120, and then the effects of p120 on NF-κB signaling pathway were detected.Results(1) LPS induced the activation of NF-κB signaling in bronchial epithelial cells (BECs) by transient transfection and luciferase reporter assay. Meanwhile, IL-8, a proinflammatory factor, which is the target gene of NF-κB was also increased significantly after LPS treatment.(2) Western blot showed that p120 was rich in BECs, but rapidly reduced by LPS as early as 15 min.(3) It was found that IκBαwas rapidly phosphorylated and degradated by LPS from 15 to 30 min.(4) The translocation of p65 from cytoplasm to nucleus after LPS treatment was confirmed by western blot and immunofluorescence. (5) After cells transfected with pEGFPp120 followed by LPS treatment, we found the activaity of NF-κB induced by LPS was partially blocked. Data also showed that IL-8 production in response to LPS was partly, but not completely down-regulated by over-expression of p120.(6) Inhibition of p120 by siRNA significantly enhanced the LPS-induced NF-κB activity, promoted LPS-induced p65 nuclear translocation and elevated IL-8 production.Conclusions(1) LPS induced the up-regulation of p120 in BECs.(2) LPS induced NF-κB activation and IL-8 production.(3) The activation of NF-κB induced by LPS was accompanied with IκBαdegradation and p65 nuclear translocation in BECs.(4) The activation of NF-κB signaling was partially inhibited by over-expression of p120, but could be significantly promoted by knocking down of p 120.These data strongly suggest that NF-κB activation is one of the cytoplasmic-nuclear signaling pathways involved in airway epithelial cells in response to LPS, and p120 is able to inhibit this activation. BackgroundNumerous studies showed that cigarette smoke have various injurious effects on epithelial cells, which may contribute to the development of lung diseases including chronic obstructive pulmonary disease (COPD) and bronchogenic lung cancer. After injury, the airway epithelium initiates a wound repair process to keep normal function, which requires spreading, migration, and eventually proliferation into the injured area. However, the specific molecular mechanisms involved in the injury and repair of airway epithelium induced by smoking have not been fully understood. Studies for cell adhesion are very meaningful due to its performance in the initial stage of wound healing.IQ domain GTPase-activating protein 1 (IQGAP1) is a multifunctional protein and could interacts with cytoskeleton, cell adhesion complex and microtubule associated proteins (MAPs). Therefore, IQGAP1 was considered to play roles in cell adhesion, polarization and migration. It has also been confirmed that IQGAP1 mediated the transcriptional activation ofβ-catenin/Tcf signaling and the expression of downstream target gene involved in cell proliferation. All the studies showed an important role of IQGAP1 in injury repair. Due to its highly expression in lung tissue, we speculated that IQGAP1 may regulate the process of airway epithelial injury and repair, and we tried to explore its possible mechanism.ObjectiveTo determine the dynamic expression of IQGAP1 and the effects of IQGAP1 on cell adhesion complex during the injury and repair process following CSE exposure, and to provide more evidences for further exploring the mechanism of injury and repair.MethodsIn this study, we established a model of injury and repair of airway epithelia by CSE in vitro. Firstly, we used the phase contrast microscope to observe the morphological changes of bronchial epithelial cells (BECs) after CSE treatment. Furthermore, MTT analysis was used to reveal the changes of cell viability after CSE treatment. Then, the following experiments were performed:Western blot, confocal laser scanning, co-immunoprecipitation. We aimed to observe the expression and localization of IQGAP1, and its impact on cell adhesion complex. Finally, transient transfection and isolation of cytoplasmic and nuclear proteins was used to detect the effects of IQGAP1 onβ-catenin.Results(1) Under phase contrast microscope, cells display morphological changes after CSE treatment, including widened cell-cell interspaces, more widely flattened appearance and more dead cells, compared with control cells, which showed a classic cobblestone-like epithelial morphology that was three-dimensional, slightly raised and closely adherent.(2) MTT results showed that low concentrations of CSE can promote cell proliferation, while high concentrations lead to a dose-dependent reduction of cell viability.(3) Western blot and immunofluoresence showed that CSE induced a time-dependent up-regulation of IQGAP1 in the airway epithelial cells and its positioning signal translocated from the cell-cell junctions to the whole cytoplasm, respectively. More meaningfully, we discovered that IQGAP1 combined withβ-catenin by immunofluorescence and Western blot. Such integration had become stronger after CSE stimulation, as well as the combination betweenβ-catenin and a-catenin was sharply reduced.(4) Over-expression of IQGAP1 by transient transfection not only increased the accumulation ofβ-catenin in the cell, but also promoted the process of its nuclear translocation. After the expression of IQGAP1 was knocked down by small interfering RNA(siRNA), the accumulation and nuclear translocation of P-catenin were reduced.Conclusions(1) CSE induced up-regulation of IQGAP1 in vitro of airway epithelial cells.(2) CSE attenuated the combination betweenβ-catenin and a-catenin, but enhanced the association between P-catenin and IQGAP1.(3) over-expression of IQGAP1 promoted the accumulation and nuclear translocation ofβ-catenin.The results suggest that IQGAP1 may weakens cell adhesion by binding to P-catenin and leading to the release of a-catenin from adhesion complex. Moreover, IQGAP1 may plays roles in cell proliferation by promoting the nuclear translocation ofβ-catenin.