Tiotropium Inhibits Methacholine-induced Extracellular Matrix Production Via ?-catenin Signaling In Human Airway Smooth Muscle Cells

BackgroundAirway remodeling is an important feature of chronic inflammatory airway diseases such as Chronic Obstructive Pulmonary Disease?COPD?and correlates with disease severity and irreversible airflow limitation.It is described as the structure changes in the airway wall caused by repeated injury and repair,including airway epithelial cell hyperplasia,squamous cell and goblet cell metaplasia,reticular basement membrane?RBM?thickening,peribronchial fibrosis and angiogenesis.Airway remodeling exists in small airways prior to the emphysematous destruction in COPD and induces clinical symptoms like shortness of breath at an early stage of COPD.However,currently there is no effective therapy for relieving or reversing airway remodeling.Extracellular matrix?ECM?alteration plays a key role in the process of airway remodeling,which is likely to contribute to persistent tissue injury and airflow obstruction in COPD patients.The ECM is a complicated network of macromolecules containing collagens,elastic fibers,proteoglycans,fibronectin and tenascin,and the content of collagens,elastic fibers and fibronectin is associated with the decline in the forced expiratory volume in one second?FEV₁?.It has been reported that there is altered ECM composition in the airways of COPD patients who have mild airflow limitation,which suggests that it is necessary to prevent abnormal ECM deposition at the early stage of COPD.It has been suggested that ECM is mainly produced by fibroblasts,but many recent studies have shown that airway smooth muscle cells?ASMCs?may play a crucial role in producing ECM when exposed to allergens,cigarettes and environmental pollution.ASMCs are the main structure cells in small airways,which are subjected to the vagus nerve and contribute to bronchoconstriction.The increased cholinergic tone is the primary reversible element of airflow limitation in COPD.Hence,tiotropium,the first long-acting inhaled muscarinic antagonist introduced to the market,has been used as a bronchodilator in COPD for more than 10 years.However,it has been suggested that mechanical forces caused by bronchoconstriction may play a crucial role in airway remodeling in asthma.Grainge et al.have found that bronchoconstriction without inflammation induced airway remodeling in asthma patients.Furthermore,in recent years,several studies have indicated that anticholinergics may have effects beyond bronchodilation.For example,Pera et al.have found that tiotropium inhibited airway remodeling including ECM deposition in a guinea pig model of COPD.These findings suggest that bronchodilators may prevent airway remodeling.However,there is not enough evidence for the relationship between ASMCs relaxation and airway remodeling,and the mechanisms involved remain unclear.The constriction and relaxation of ASMCs are associated with remodeling of the cytoskeleton.?-catenin is a protein that connects cadherins to the cytoskeleton at adherens junctions where it functions as a membrane-bound protein and influences cell stabilization and transmission of mechanical forces.However,cytosolic?-catenin plays a central role in the canonical Wnt/?-catenin signaling pathway.When it is rescued from glycogen synthase kinase3??GSK3??-mediated phosphorylation and proteasomal degradation,it translocates into the nucleus and regulates T-cell factor?TCF?/Lymphoid enhancer factor?LEF?-mediated gene transcription.Recently,it has been suggested that mechanical forces within the airway wall can activate?-catenin signaling.Activation of?-catenin signaling?in WNT-dependent and–independent manners?has a regulatory function in airway remodeling involving ASMCs proliferation,epithelial-to-mesenchymal transition,myofibroblast differentiation and ECM production.Based on the aforementioned clues,we hypothesized that tiotropium inhibits ECM production through?-catenin signaling in ASMCs.ObjectiveTo investigate whether tiotropium can inhibit methacholine-induced extracellular matrix production in airway smooth muscle cells and the mechanism involved.MethodsPart One:Primary culture and identification of airway smooth muscle cellsHuman ASMCs?HASMCs?were isolated from healthy segments of the lobar or main bronchus obtained from three different lung resection donors after giving informed consents.Primary cultured HASMCs from passages 2 to 10 were used for the experiments and the experiments were triplicate in the three cell lines.Cells were incubated in Dulbecco's modified Eagle's medium?DMEM?with 10%fetal bovine serum and antibiotics.Cells were washed twice with phosphatebuffered saline?PBS?,and then serum starved in DMEM with antibiotics for 12h.Subsequently,cells were stimulated with methacholine?10mM?in serum-free medium supplemented with antibiotics for 24h.Tiotropium?10mM?was added 30min before the addition of methacholine.Cells were identified by immunofluorescence with specific staining of smooth muscle-specific alpha actin?a-SMA?and myosin heavy chain?MHC?.Part Two:Western blot analysisWhole cell lysates were extracted using the radioimmunoprecipitation assay?RIPA?lysis buffer containing protease inhibitors and phosphatase inhibitors.Nucleoproteins were extracted using Nuclear-Cytosol Extraction Kit according to the manufacturer's instructions.Protein concentration was detected by the BCA Protein Assay Kit.All the lysates were stored at–80°C until further use.Western blotting was conducted following standard procedures.Equal amounts of protein?15–25mg/lane?were electrophoresed in 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?and transferred to polyvinylidene fluoride?PVDF?membranes.The membranes were blocked with 5%BSA for 2h,and then incubated with specific primary antibodies at 4°C for at least 12h.Subsequently,the blots were incubated with horseradish peroxidase?HRP?-conjugated secondary antibodies for 1h at room temperature.The proteins of interest were detected by enhanced chemiluminescence reagents,and the band intensities were quantified using the Image J software.The expression of the target proteins was normalized against the loading control,?-actin or Lamin B1.Part Three:Isolation of mRNA and real-time PCR analysisTotal mRNA was extracted using MiniBEST Universal RNA Extraction Kit according to the manufacturer's protocol.The mRNA was quantified by a NanoDrop Spectrophotometer.For reverse transcription,1mg of total mRNA per sample was used with PrimeScript RT reagent Kit with gDNA Eraser and c DNA was stored at–20°C until further use.The gene-specific primers were obtained from Sangon Biotech.Quantitative real-time PCR was performed with SYBR Premix Ex Taq II and Roche Light Cycler 480 instrument II.The target gene levels were quantified with the 2^?–??ct?relative quantification method,which was normalized to GAPDH.Part Four:?-catenin S33Y mutant transfectionThe active?-catenin mutant,S33Y-?-catenin,is resistant to GSK3-mediated phosphorylation and proteasomal degradation because of aserine-to-tyrosine substitution at position 33.The adenovirus packaging was conducted by a professional company,and the transduction efficiency was measured by green fluorescent protein?GFP?fluorescence using a fluorescence microscope.Cells were incubated in DMEM with 10%FBS.The recombinant adenovirus was directly transfected into HASMCs at 50%confluence in six-well cluster plates for 48h[multiplicity of infection?MOI?=100].A GFP expression vector was used as a negative control.Consecutively,the medium was replaced with serum-free DMEM,followed by tiotropium stimulation,which was added 30min before the addition of methacholine.Transfected cells were harvested for total protein or mRNA extraction after 24h.Part Five:b-catenin-siRNAtransfectionA specific double-stranded small interfering RNA?siRNA?against theb-catenin transcript or anegative control was transfected into HASMCs at a final concentration of 90nM when cells were 50%confluent in six-well cluster plates using Lipofectamine 2000 transfection reagent according to the manufacturer's instructions.Cells were cultured in serum-free DMEM without any supplements for6h.Next,cells were washed once with PBS,and then incubated in DMEM with 10%FBS for another 42h.Subsequently,the medium was replaced with serum-free DMEM and stimulated with tiotropium,which was added 30min before the addition of methacholine.Transfected cells were harvested for extraction of total protein or mRNA after 24h.Part Six:ELISAAccording to the manufacturer's protocol of ELISA Kit.Part Seven:Statistical analysisAll quantitative data are presented as mean±SD and analyzed using SPSS v.16.0.Differences between two groups were evaluated by the independent t-test.Multiple comparisons were analyzed by one-way analysis of variance?ANOVA?,followed by Student–Neuman–Keuls test with equal variances determined by the homogeneity of variance test.Differences were considered to be statistically significant when P<0.05.ResultsPart One:Cell culture and identificationPrimary cultured HASMCs were fusiform when observed under an inverted light microscope and showed the typical“hill and valley”pattern when confluent.We used the second generation of cells for identification.Cells expressed smooth muscle?-actin??-SMA?and myosin heavy chain?MHC?,the contractile phenotype marker proteins and the contractile fibrils were visible when magnified at 400 times.Part Two:Methacholine induced extracellular matrix production in HASMCsFirst,we investigated the effect of methacholine on ECM production.We stimulated cells with an increasing concentration of methacholine from 0.1mM to10mM for 24h.The expression of collagen I reached the maximum at 10mM.Thus,we performed the following experiments with 10mM methacholine.Then we stimulated cells with 10mM methacholine for different time points?0⁴8h?.The expression of collagen I reached the maximum at 24h.Furthermore,real-time PCR showed that the gene expression of collagen I,fibronectin,versican,Laminin?2 was increased in HASMCs exposed to 10mM methacholine while decorin was decreased.Part Three:Tiotropium inhibits methacholine-induced ECM expressionThen,we investigated whether tiotropium can inhibit methacholine-induced ECM production in HASMCs.We stimulated HASMCs with increasing concentrations of tiotropium?0.1mM to 100mM?.Western blotting revealed that tiotropium decreased collagen I protein expression from 10mM.Accordingly,we used tiotropium at 10mM for the following experiments.Besides,real-time PCR showed that the gene expression of collagen I,fibronectin and versican was decreased in HASMCs exposed to 10mM tiotropium.These data revealed that tiotropium inhibited ECM production in HASMCs.Part Four:Tiotropium inhibits?-catenin signaling in HASMCsNext,we investigated whether tiotropium affected?-catenin signaling.HASMCs were stimulated with different tiotropium concentrations?0.1mM to100mM?.Both total?-catenin and active?-catenin protein expression were significantly decreased from 10mM,which is in accordance with the effect on ECM production.Moreover,tiotropium decreasedb-catenin mRNA abundance in HASMCs.GSK3?phosphorylation is necessary for the activation of?-catenin signaling.Western blotting demonstrated that tiotropium markedly inhibited GSK3?phosphorylation in HASMCs.These data indicated that tiotropium suppressed?-catenin signaling by preventing GSK3?phosphorylation.Part Five:Tiotropium inhibits the transcription activity of active?-cateninThen,we investigated the effect of tiotropium on the transcription activity of active?-catenin.Western blot analysis showed that tiotropium reduced the expression of active?-catenin.Non-phosphorylated?-catenin translocates from the cytoplasm to nucleus and interacts with TCF/LEF to start target genes transcription.Hence,we examined the amount of active?-catenin in the nucleus.With tiotropium pre-incubation,the abundance of active?-catenin that translocated into the nucleus was also decreased.These data showed that tiotropium decreased the amount of active?-catenin and suppressed its transcription activity.Part Six:Overexpression of?-catenin suppresses the effect of tiotropium on ECM productionTo further investigate the role of?-catenin in the tiotropium-induced inhibition of ECM expression in HASMCs,we examined the negative effect of tiotropium on the expression of collagen I protein when?-catenin is overexpressed.To this end,we used adenoviruses carrying a constitutively active?-catenin mutant?S33Y-?-catenin?.The mutant has a serine-to-tyrosine substitution at position 33,rescuing?-catenin from GSK3?-mediated phosphorylation and proteasomal degradation.Primary cultured HASMCs were transfected with the S33Y-?-catenin mutant,while control cultures were transfected with GFP.A satisfactory infection efficiency was obtained at MOI=100,which was measured by green fluorescence under fluorescence microscopy.The abundance ofb-catenin mRNA was elevated as evidenced by real-time PCR and the expression of total?-catenin was markedly increased.Furthermore,transfection with the S33Y-?-catenin mutant increased the basal level of active?-catenin.In the control cells,tiotropium reduced the methacholine-induced expression of active?-catenin and collagen I,as described before.However,tiotropium did not decrease the methacholine-induced production of active?-catenin and collagen I when cells were transfected with the S33Y-?-catenin mutant.In fact,the expression level was higher than it was upon stimulation with methacholine alone.These data revealed that?-catenin played a crucial role in the suppression of methacholine-induced collagen I expression by tiotropium in HASMCs.Part Seven:The effect of?-catenin silencing on the tiotropium-induced inhibition of ECM productionWe investigated the effect of?-catenin silencing on the suppressive effect of tiotropium on collagen I expression in HASMCs.Cells were transfected with specificb-catenin siRNA and the optimal reduction in?-catenin expression was achieved with 90nMb-catenin siRNA.Silencing of?-catenin reduced the basal level of total?-catenin.As mentioned previously,tiotropium inhibited the methacholine-induced expression of total?-catenin and collagen I.When transfected withb-catenin siRNA,the methacholine-induced total?-catenin and collagenI protein expression was downregulated.Furthermore,the expression level of total?-catenin and collagen I was much lower when cells were treated with tiotropium together with?-catenin silencing compared with the effect of tiotropium alone.These data indicated that?-catenin was sufficient for the tiotropium-induced inhibition of collagen I production.Part Eight:The effect of tiotropium on fibronectin secretionFinally,we detected the secretion of fibronectin with tiotropium.Methacholine has no effect on fibronectin secretion and there is no significant difference between the groups with or without tiotropium.These data indicated that tiotropium has no effect on fibronectin secretion.ConclusionTiotropium inhibits methacholine-induced extracellular matrix production via?-catenin signaling in human airway smooth muscle cells and it has no effect on fibronectin secretion.