Role Of Glycogen Synthase Kinase 3 And IQ Domain GTPase-activating Protein 1 In The Toxic Injury Of Airway Epithelia

PartⅠBleomycin-induced nuclear factor-κB activation in humanbronchial epithelial cells involves the phosphorylation ofglycogen synthase kinase 3βBackground:Bleomycin(BLM) is an effective chemotherapeutic antibiotic which has beenused successfully to treat a wide variety of malignancies such as squamous cellcarcinoma,sarcoma and lymphoma.However,the clinical application of the drug islimited by its adverse side effects,especially severe lung injury and fibrosis.Epithelialcells are known to be a main target of BLM lung toxicity.Exposure of cells to BLMresults in diverse cellular responses including apoptosis,cell death and secretion ofpro-inflammatory cytokines,which contribute to the development of lung injury andfibrosis induced by BLM.Nuclear factor-κB(NF-κB) regulates a large number of genesinvolved in cell survival,differentiation,proliferation,apoptosis and inflammation andis also considered to be closely correlated with BLM lung toxicity.NF-κB is normally sequestered in the cytoplasm of resting cells by inhibitor ofNF-κB(IκB) and remains transcriptionally inactive.Stimulation by cytotoxic triggerssuch as BLM and tumor necrosis factorα(TNFα) induces the ubiquitylation anddegradation of IκB.The loss of IκB exposes the nuclear localization signal sequence onNF-κB,resulting in the nuclear translocation of NF-κB and transcriptional activation ofNF-κB target gene promoters.Glycogen synthase kinase 3β(GSK3β) is a ubiquitousand important serine/threonine kinase.In contrast to most other kinases,GSK3βisconstitutively active in resting cells and can be inactivated by phosphorylation on anN-terminal serine residue(Ser~9) through a PI3K(phosphatidylinositol 3-kinase)/Akt(also called protein kinase B,PKB)-dependent mechanism.Recent studies revealed that GSK3βmight be involved in the control of various signaling pathways that activateNF-κB,and the effect of GSK3βon the regulation of NF-κB raised an interest in thepathophysiology of the diseases in which NF-κB activation is involved.However,theexact mechanisms are still unclear.Therefore,our present studies focus on the changesof GSK3βactivity and the effects of GSK3βon NF-κB signaling during the toxic lunginjury induced by BLM.ObjectiveTo investigate the changes of GSK3βactivity and the effects of GSK3βon NF-κBsignaling during the toxic lung injury induced by BLM.MethodsIn this study,we treated the cells with BLM to establish the lung toxic injury modelin vitro.Firstly,we used the phase contrast microscope and fluorescence staining toobserve the morphological changes of bronchial epithelial cells(BECs) after BLMtreatment.Furthermore,MTT analysis was used to reveal the changes of cell viabilityafter BLM treatment.Then the following experiments were performed:Western blot,confocal laser scanning,co-immunoprecipitation,transient transfection,luciferasereporter assay and so on.We aimed to study the active status of NF-κB signaling,theactivity changes of Akt/GSK3βpathway and its effects on NF-κB signaling after BLMtreatment.ResultsUnder phase contrast microscope,treatment with BLM made cells displaymorphological changes,including widened cell-cell interspaces,more widely flattenedappearance and numerous dead cells,compared with control cells,which showed aclassic cobblestone-like epithelial morphology that was three-dimensional,slightlyraised and closely adherent.In addition,nuclear morphological changes were observed under fluorescence microscope by Hoechst 33258 staining.Results showed that BLMcaused nuclear condensation and cell apoptosis.MTT analysis revealed adose-dependent reduction of cell viability after BLM treatment.Then,we further confirmed that BLM induced the activation of NF-κB signaling inBECs by transient transfection and luciferase reporter assay.Furthermore,theexpression of IκBαreduced and almost disappeared within 2 h after BLM treatment.Inthe same time,both the data from immunofluorescence staining and Western blot ofnuclear extract revealed that incubation with BLM in BECs caused a striking shift ofp65(the subunit of NF-κB) towards the nucleus.These results are consistent with theprevious experiments in vivo which have been published.Subsequently,we found thatphosphorylation levels of Akt and GSK3βrapidly increased in 0.5 h after BLMtreatment and became higher at 2 h.However,pretreated with LY294002,whichinhibited BLM-induced phosphorylation of Akt and GSK3β,the transcriptionalactivation of NF-κB in response to BLM was also significantly reduced in adose-dependent manner.Thus we suppose that BLM-induced NF-κB activation must beclosely associated with the active status of GSK3β.So cells were transiently transfectedwith a stable mutant of GSK3β(S9A) which is constitutively active followed by BLMtreatment.The results showed that continuous activation of GSK3βled to a partial butsignificant inhibition of BLM-induced NF-κB activation,protected endogenous IκBαfrom degradation and blocked the nuclear translocation of p65 subunit.To investigatethe possible cause of this phenomenon,a co-immunoprecipitation assay was used andthe results showed that GSK3βwas indeed able to be co-precipitated with IκBαin BECs.Moreover,the co-precipitated proteins decreased after BLM treatment.Importantly,there was no phosphorylated GSK3βto be detected in IκBαprecipitates,indicating thatIκBαmight be preserved by a mechanism that is dependent on direct GSK3βbinding.These data further demonstrated that GSK3βaffected BLM-induced NF-κB activationthrough an interaction with IκBα. Conclusions(1) BLM induced the morphological changes and reduction of cell viability in BECs.(2) BLM induced NF-κB activation,accompanied with IκBαdegradation and p65nuclear translocation in BECs.(3) BLM induced the phosphorylation of Akt and GSK3βin BECs.(4) GSK3βmight negatively regulate BLM-induced NF-κB activation in BECs by amechanism that is dependent on the interaction of GSK3βand IκBα. PartⅡEffects of IQ domain GTPase-activating protein 1 on APCprotein andβ-catenin/Tcf signaling during the toxic injury ofairway epithelia after formaldehyde exposureBackground:Formaldehyde(FA) is a ubiquitous chemical pollutant,which can induce chronictoxic respiratory disease of animals or human after long-term and low-concentrationtouch.It even leads to gene mutation and increases the probability of some malignanttumours in human.It has been confirmed that IQ domain GTPase-activating protein 1(IQGAP1) isalways over-expressed in most tumours with unclear mechanism.IQGAP1,animportant effector of Rho GTPase family including Cdc42 and Racl,has many proteinbinding sites,not only regulating cell skeleton and affecting cell adhesion,but alsopossibly mediating the transcriptional activation ofβ-catenin/Tcf signaling and theexpression of downstream target gene involved in cell proliferation,such as cyclin D1and c-myc.On the other hand,active Cdc42 and Racl can promote APC proteinstabilizing the plus end of microtubule in the leading edge of migrating cells throughbinding with IQGAP1 and play key roles in cell polarization and migration.Nevertheless,a great quantity of recent studies demonstrated that APC protein alsolocated in the nuclei where it functions in regulating the spindle and inhibits cellproliferation by interfering with the activation ofβ-catenin/Tcf signaling.Therefore,inthe present study,we established the toxic injury model of airway epithelia afterformaldehyde exposure in vivo and in vitro,and observed that the dynamic changes ofIQGAP1 during the process of injury and repair.We further studied the effects ofIQGAP1 on APC protein andβ-catenin/Tcf signaling in vitro by using transfection andsmall interfering RNA technique.We aim to understand the injury and repair process of airway epithelia following formaldehyde exposure,providing a novel idea in elucidatingthe potential tumourigenicity mechanism of formaldehyde.ObjectiveTo determine the dynamic changes of IQGAP1 and the effects of IQGAP1 onAPC protein andβ-catenin/Tcf signaling during the toxic injury of airway epitheliafollowing formaldehyde exposure.MethodsIn this study,we established the toxic injury model of airway epithelia afterformaldehyde exposure in vivo and in vitro.We first observed the pathological changesof murine airway epithelia following formaldehyde inhalation under light microscopeafter H&E staining.Then we detected the location and expression of IQGAP 1 by usingimmunofluorescence staining and Western blot.In our experiment in vitro,we used thephase contrast microscope and MTT method to observe the morphological changes ofbronchial epithelial cells(BECs) after formaldehyde exposure and the effect of IQGAP 1over-expression on cell proliferation.Pull down assay was used to detect the activity ofCdc42 and Racl.Immunofluorescence staining and confocal laser scanning microscopewere used to determine the subcellular location of APC andβ-catenin protein.Thefollowing experiments were performed:Western blot,co-immunoprecipitation assay,transient transfection,small interfering RNA,luciferase reporter assay and so on.Weaim to study the effects of IQGAP1 on APC protein andβ-catenin/Tcf signaling,understand the injury and repair process of airway epithelia following formaldehydeexposure,providing a novel idea in elucidating the potential tumourigenicitymechanism of formaldehyde.ResultsIn the process of formaldehyde inhalation,the murine airway epithelia experiencedthe dynamic course from increased number of goblet cells,lodgings and losses of cilia, loose contacts between cells,infiltrations of inflammatory cells,shedding of necroticcells into the airway lumen to the re-appearance of cilia,tighter cell-cell junction,hyperplasia of basal layer cells,increased cellular layer and infiltration of chronicinflammatory cells.The expression of IQGAP 1 showed a corresponding tendency withthe progression of injury and repair.A strong signal of IQGAP 1 diffused in the wholecytoplasm in the early stage of severe injury and located around the plasma membraneagain in the end stage of repair,implicating that IQGAP 1 plays an important role in theinjury and repair of airway epithelia following the toxic stimulation from formaldehyde.Under phase contrast microscope,formaldehyde exposure made cells lose itsnatural classic cobblestone-like epithelial morphology and display widened cell-cellinterspaces,more widely flattened appearance,even cellular condensation andnumerous dead cells when exposed to higher concentration.MTT analysis revealed thatlow-concentration formaldehyde promoted cell proliferation and high-concentrationformaldehyde induced a dose-dependent reduction of cell viability.Then,pull downassay was used to detect the activity of Cdc42 and Racl and the results showed adifferent alteration after formaldehyde exposure.After that,we further determined theexpression and location of their effectors,IQGAP 1.The data from Western blot analysisshowed that formaldehyde induced the time-and concentration-dependentover-expression of IQGAP1 in the airway epithelial cells.The fluorescent signal alsotranslocated from the cell-cell boundary and circum-nuclei to the whole cytoplasm.Importantly,we found that IQGAP1 over-expression stimulated cell proliferation andelevated the cell viability significantly,which might be achieved through increasing theaccumulation ofβ-catenin in the cell and promoted the process of its nucleartranslocation.After the expression of IQGAP1 was knocked down by small interferingRNA(siRNA),not only formaldehyde-inducedβ-catenin translocation,but also theactivation ofβ-catenin/Tcf signal and the expression of its downstream target genecyclin D 1 were inhibited,further confirming a key role of IQGAP 1 in cell proliferation.Finally,when we detected the expression and location of APC protein in airway epithelial cells,we surprisingly found that formaldehyde not only induced the increaseof APC protein but also resulted in a striking translocation from diffusion in the wholecytoplasm to accumulation in the nuclei and one side of cytoplasm.To understand thecause of this phenomenon,co-immunoprecipitation assay was used to detect the relationof IQGAP 1 and APC.The results showed that they could be precipitated each other andthe interaction was enhanced after formaldehyde exposure.So we proposed that the sideof APC accumulation might signify the direction of cell migration and IQGAP1participated in cell migration through binding with APC.Importantly,the nucleartranslocation of APC protein with unknown mechanism might function as a terminationsignal in the protein network under complicate regulations to inhibit the activation ofβ-catenin/Tcf signaling and prevent the excess proliferation of epithelial cells in therepair process after injury.Conclusions(1) Formaldehyde induced the over-expression of IQGAP 1 in vivo and in vitro.(2) Over-expression of IQGAP1 resulted inβ-catenin accumulation and nucleartranslocation,activatedβ-catenin/Tcf signaling,elevated expression levels of cyclinD 1 and promoted cell proliferation in airway epithelial cells.(3) The enhanced interaction of IQGAP1 andβ-catenin and APC after formaldehydeexposure might be correlated with cell migration and cell adhesion.(4) Formaldehyde exposure up-regulated the expression of APC protein in airwayepithelial cells and made it accumulate in one side of cytoplasm and the nuclei withthe tighter interaction of IQGAP1 and APC in the cytoplasm,which could playimportant roles in signifying the direction of cell migration and preventing theexcess proliferation of epithelial cells.