The Role Of TROP2 In Airway Remodeling In Chronic Obstructive Pulmonary Disease

Background:Chronic Obstructive Pulmonary Disease(COPD)is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases.As airway epithelium is the primary target of inhaled harmful particles,abnormal tissue repair has become a primary focus in understanding the process of airway remodeling.Basal cells(BCs)include multipotent stem/progenitor progenitor cells of bronchial airway epithelium and make a major contribution to the regeneration of bronchial epithelium.These cells play a key role in the maintenance of the normal airway epithelial architecture through their capacity to self-renew,differentiate into ciliated and secretory cells,and establish interactions with mesenchymal cells.Abnormalities in the number and function of BCs may therefore severely affect the regeneration of injured airway epithelium such as in smoking.Basal cell hyperplasia caused by smoking has occurred long before lung function declines,indicating that the development of COPD may begin with these cells.However,little is known about the molecular mechanisms underlying the abnormal biological behavior of BCs in COPD.Trophoblast cell surface antigen 2(TROP2),is a type I transmembrane glycoprotein with low to no expression in normal tissues.It provides crucial signals for cells with requirements for self-renewal,survival,and invasion.Although TROP2 has been reported to be highly expressed in various types of epithelial cancers,including colorectal cancer,pancreatic cancer,and oral squamous-cell carcinoma,TROP2 expression has also been found in stem cells in various tissue types.In human and mouse prostate,the TROP2 expressing subpopulation of BCs possess stem cell capacities such as self-renewal,regeneration and differentiation.Undifferentiated oval cells express TROP2 shortly after activation due to liver injury.TROP2 is also enriched in endometrial-regenerating cells in a dissociated cell tissue recombination assay.These results indicate that TROP2 might play an important role more generally in the regulation of the growth and regeneration of stem cells as well as serve as a molecular marker of them in various adult tissues.Here,protein expression of TROP2 was specifically examined in the development of COPD in smokers.TROP2 was furthermore overexpressed in normal airway BCs in vitro in order to identify potential biological and molecular pathways regulated by the protein.Methods:1.The expression of TROP2 in airway BCs from COPD patients1.1 PatientsProtocols for recruitment of patients and healthy,nonsmoking individuals as well as the collection of tissue samples and the analysis of patient data were approved by the ethics committee at Qilu Hospital of Shandong University.Written informed consent was obtained from each individual for participation in the study.Lung tissue samples were obtained from patientsat Qilu Hospital of Shandong University during lobectomy or pneumonectomy performed for medical reasons,including lung tumor,pneumatocele and pulmonary cyst.The cohort consisted of smokers with COPD(n = 24).smokers without COPD(n = 24),and nonsmokers(n = 24)as controls.The diagnosis of COPD was made according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease(GOLD).1.2 Immunohistochemical staining was used to detect the expression of TROP2 in lung tissues,and the expression of TROP2 in airway basal cells was analyzed by immunofluorescence staining of Cytokeratin 5 and TROP2.1.3 Spearman rank correlation was used to examine the relationship between TROP2 expression and FEVi%.1.4 Inmunohistochemistry detecting Ki67 expression in sections of paraffin embedded lung tissues from patients as indicated.1.5 Correlation of the level of TROP2 protein expression with Ki67 in COPD patient samples as determined by immunohistochemistry.2.Effect of TROP2 on airway basal cell proliferation2.1 Isolation and culture of BCs The primary epithelial cells were derived from healthy donors and COPD patients(FEV:/FVC<70%,FEV1%≥500%predicted).All the donors were off corticosteroid use in recent month.Airway epithelial cells were obtained by gently brushing the airway epithelium using flexible bronchoscopy as previously described.Cells were collected and cultured in a T25 cell culture flask with BEGM in 5%C02 in a humidified chamber at 37℃.2.2 Cyto-immunofluorescence for the identification of BCs BCs were incubated with primary antibody against Cytokeratin 5 and p63 overnight and incubated with TRITC conjugated anti-rabbit IgG and FITC conjugated anti-mouse IgG at room temperature for 30 min.Cells were and visualized with confocal fluorescence microscopy.2.3 Transfection BCs were transfected with pcDNA3.1-TROP2 or siRNA sequence using LipofectamineTM 2000 Transfection Reagent according to the manufacturer’s instructions.Cells transfected with empty vector or scrambled siRNA were considered as negative control.At 48 h,cells were trypsinized,and experiments were performed.2.4 The experiment was divided into treatment group(transfected with pcDNA3.1-TROP2 or siRNA),negative control and blank control cells.The effect of TROP2 on airway basal cell proliferation and repair capacity were detected by CCK-8,cell cycle assay,and scratch test.2.5 Western blot analysis was performed on cell lysates prepared from transfected(pcDNA3.1 and pcDNA3.2-TROP2)and control cells 48 h after transfection and inhibition of the ERK/MAPK pathway with U0126 as indicated(+/-).O.D.450 nm of CCK-8 was plotted as a function of time to assess viability/proliferation of pcDNA3.1-TROP2-BCs treated with the ERK/MAPK pathway inhibitor U0126.3.Effects of TROP2 on EMT and the secretion of inflammatory cytokines in airway basal cells3.1 Immunofluorescence staining of Cytokeratin 5 and TROP2 was performed on lung tissues from COPD patients and controls.3.2 Airway basal cells were transfected with pcDNA3.1-TROP2 or siRNA.The changes of EMT markers were detected by Western Blot method.3.3 Cells were transfected with pcDNA3.1-TROP2.The supernatants were harvested 48 h later,and the levels of IL-6,IL-8,and IL-1β were quantified in the supernatants using an ELISA kit according to the manufacturer’s instructions.3.4 Airway basal cells were precubated with IL-6,IL-8,and IL-1β neutralizing antibody,the expression of E-cadherin and vimentin in pcDNA3.1-TROP2-BC was assessed.3.5 ERK1/2 specific inhibitor U0126 was used to pre-treat the cells.After TROP2 overexpression,EMT markers in airway basal cells were detected by Western blot and was used to detect the changes of inflammatory cytokines were detected by Elisa.Results1.Patient characteristicsThe forced expiratory volume in first second percentage(FEV1%)of predicted and the FEV1/forced vital capacity(FVC)ratios were significantly reduced in smoking patients with COPD compared to healthy controls(P<0.05 for both comparisons).The values in the FEV1%of predicted and the FEV1/FVC ratios were statistically similar between smokers without COPD and nonsmokers(P = 0.41 and P = 0.30,respectively).Although the smoking index in smokers with COPD seems to be a little higher than that in smokers without COPD,there was no statistical significance(P = 0.10).2.TROP2 expression is elevated in airway BCs in COPD lung tissue samplesTROP2 expression was significantly increased in COPD relative to nonsmoker and smoker controls as well as in smoker controls relative to nonsmokers(P<0.05 for all).Immunofluorescent double staining showed that TROP2 staining was coincident with cytokeratin 5,indicating that expression of the protein originated in part from airway BCs.3.TROP2 expression correlates with the degree of airflow obstruction in COPD patientsThe analysis revealed a significant inverse correlation between FEVi%and TROP2 expression(r =-0.53,P<0.01).4.Increased TROP2 expression correlates with a high airway proliferative index in COPD tissue samplesThe density of Ki67 positive cells was significantly higher in hyperplasic epithelium of COPD patients than that in normal airway epithelium(P<0.05).Analysis of immunostaining for the two proteins revealed a statistically significant correlation betxween increased TROP2 expression and Ki67 positive cells in COPD lung tissue samples(r ＝0.878,P<0.01).5.TROP2 promotes proliferation and wound closure in BCs in vitroBasal cells were transfected with an expression vector for TROP2,pcDNA3.1-TROP2.At 48 h post-transfection,mRNA and protein levels of TROP2 were increased by～-2.5 fold in BCs,as assessed by real-time PCR and Western blots analysis.In the CCK-8 assay,O.D.450 values were increased in pcDNA3.1-TROP2-BCs,indicating that cell viability was significantly improved in these cells relative to controls(P<0.05).The percentage of cells in S and G2/M was significantly increased in pcDNA3.1-TROP2-BCs relative to control cells(P<0.05).At 24 and 48 h after wounding,pcDNA3.1-TROP2-BCs displayed an increased capacity for wound closure relative to control cells(P<0.05).6.Down-regulated TROP2 decreases proliferation and wound closure in COPD-derived BCsTROP2 was down-regulated by siRNA in COPD-derived BCs.At 48 h post-transfection,the result Western blots analysis showed that mRNA and protein levels of TROP2 were decreased by-4.5 fold in BCs.Cell viability in the siRNA-TROP2-BCs was significantly inhibited(P<0.05).the percentage of cells in S was significantly decreased in siRNA-TROP2-BCs relative to control cells(P<0.05).The wound healing assay revealed that,at 24 and 48 h after wounding,siRNA-TROP2-BCs exhibited a weakened capacity for wound closure relative to control cells(P<0.05).7·Activation of ERK/MAPK signaling stimulates BC proliferationAt 48 h after transfection,protein levels of pERK1/2 were up-regulated in pcDNA3.1-TROP2-BCs.Expression of cyclin D1 was also increased significantly.Treatment with U0126 blocked the increase in pERK1/2 and inhibited TROP2 mediated BC proliferation assessed by CCK-8 assay,indicating that pERK1/2 might be involved in the signaling pathway.8.The expression of EMT in airway basal cells from COPD patientsWe observed significant immunostaining of vimentin in airway basal cells from lung tissue of COPD patients.A marked increase in the number of vimentin positive cells was observed within airway basal cells from COPD compared with controls.9.TROP2 induces an epithelial-mesenchymal transition(EMT)-like phenotype in airway BCs in vitroThe protein levels of the epithelial marker E-cadherin were significantly decreased in pcDNA3.1-TROP2-BCs(P<0.05)whereas the expression of the mesenchymal marker vimentin was increased compared to control cells(P<0.05).In contrast,down-regulation of TROP2 significantly increased E-cadherin expression and decreased vimentin expression in COPD-derived BCs(P<0.05).10.Secretion of proinflammatory cytokines is increased in BCs overexpressing TROP2The levels of levels of inflammatory cytokines IL-6,IL-8,and IL-1β were increased in the supernatants of pcDNA3.1-TROP2-BCs relative to supernatants of control cells(P<0.05 for all factors;).11.Proinflammatory cytokines were not required for TROP2-induced EMT in BCsEMT like changes in pcDNA3.1-TROP2-BCs could not be inhibited by pretreatmet with IL-6,IL-8 and IL-1β neutralizing antibody,as neither E-cadherin nor vimentin expression showed significant difference between the treated group and the control group(P>0.05).12.Effects of blocking MAPK/ERK1/2 pathway on EMT and inflammatory cytokine secretion in basal cellsWhen pretreated pcDNA3.1-TROP2-BCs with U0126,which is an ERK1/2 specific inhibitor,neither expression of EMT markers nor secretion of inflammatory factors was significantly affected(P>0.05;P>0.05).It indicated that ERK1/2 pathway might not play a role in the EMT changes or inflammatory factor secretions.Conclusion:The results demonstrate that TROP2 expression was particularly enriched in airway BCs of patients with COPD.TROP2 may play a crucial role in COPD by affecting BC function and thus airway remodeling through increased BC hyperplasia,EMT-like change,and introduction of inflammatory molecules into the microenvironment.