The Effect Of Urokinase Plasminogen Activator And Its Receptor On EMT In The Small Airways Epithelium Of COPD

BackgroundChronic obstructive pulmonary disease (COPD) is a common disease with increasing mortality and morbidity. COPD represents a major health problem. Although COPD has received increasing attention from the medical community in recent years, it is still relatively unkown or ignored by the public as well as public health and government officials.In1953, Spain DM identify the nature of COPD which might produce obstruction in these small airways. In the majority of cases, COPD is caused by cigarette smoking, which induces chronic airway inflammation, leading to irreversible airflow limitation and an accelerated decline in lung function. The narrowing of small conducting airways before the onset of emphysematous destruction may explain the increased peripheral airway resistance reported in COPD. The key effector cell in airway fibrogenesis is the myofibroblast. Small airway epithelial cells may acquire a mesenchymal phenotype and serve as an important source of fibroblasts and myofibroblasts, through a process known as epithelial-mesenchymal transition (EMT). However, the mechanism on EMT of small airway epithelial cells is currently poorly understood.Our group previously demonstrated that urokinase plasminogen activator (uPA) and its receptor (uPAR) are highly expressed in the small airway epitheliium of patients with COPD compared with normal controls. uPAR/uPA is involved in the small airway remodeling in COPD, leading to the eventual decline in lung function. However, the role of uPAR/uPA in EMT in small airways of COPD is still unknown. Our study try to clarify that uPA and uPAR are related to EMT in human small airway epithelium with COPD.ObjectiveTo explore the effect and the related mechanism of uPA and uPAR on EMT in human small airway epithelium of COPD.Methods1. Tests on human Lung tissue sections(1) Sample collectionLung tissues were obtained from78patients (non-smokers, smokers without COPD, non-smokers with COPD and smokers with COPD) following obectomy or pneumonectomy for various medical reasons. The diagnosis of COPD was made according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease.(2) The expression of EMT、uPA and uPAR in patient small airway epitheliumExpression of the epithelial marker, E-cadherin and the mesenchymal marker, vimentin, uPA and uPAR expression were assessed in small airway epithelium by immunohistochemistry.(3) Correlation analysis between the expression of EM、uPA、uPAR and lung functionThe Spearman test was used for correlation analyses. We investigated whether vimentin、uPA、uPAR expression in the small airways correlated with FEV1%of predicted and the correlations between the expression of vimentin in small airway epithelium and uPA、uPAR expression in the small airways. 2. Experiments on cell line in vitro(1) CSE-induced EMT in cultured HSAEpiCsCell morphologic phenotypes were examined under an inverted phase-contrast microscope without or with5%CSE.The expression of E-cadherin、α-catenin、N-cadherin and a-smooth muscle actin (α-SMA) in HSAEpiCs at both mRNA and protein levels were detected by qRT-PCR and Western Blot.CSE induced EMT was associated with increased cell migration as shown by the transwell assays.(2) uPAR signaling pathway is required for CSE-induced EMT in HSAEpiCsThe expression of uPAR in CSE stimulated HSAEpiCs at both mRNA and protein levels was detected by qRT-PCR and Western Blot.For transfection treatment, HSAEpiCs cells was transfected with uPAR shRNA or empty vector using Lipofectamine2000according to the instruction. After knockdown of uPAR, the expression of uPAR was verified by qRT-PCR and Western blot assays. The cell morphology was observed after uPAR gene silencing by inverted phase-contrast microscope. The expression of EMT-related markers was detected by Western blot.The expression of p-Akt in HSAEpiCs with CSE was detected by Western blot. The effect of uPAR gene silencing on Akt activation was detected by Western blot. The effect of PI3K inhibitor (LY294002) on the expression of EMT-related markers was detected by Western blot.(3) uPA is required for CSE-induced EMT in HSAEpiCs cells The expression of uPA in CSE stimulated HSAEpiCs at both mRNA and protein levels was detected by qRT-PCR and Western blot.To test whether uPA is responsible for CSE-induced EMT, we treated HSAEpiCs cells with amiloride (for uPA inhibition). The cell morphology after uPA inhibition was observed by inverted phase-contrast microscope. Cell migration after uPA inhibition was tested by the trans well assays. The expression of EMT-related markers were detected by Western blot.We overexpressed uPA in HSAEpiCs cells using Lipofectamine2000according to the instruction. The expression of uPA was verified by Western Blot. The cell morphology in uPA-overexpressing HSAEpiCs was observed by inverted phase-contrast microscope. Cell migration in uPA-overexpressing HSAEpiCs was tested by the transwell assays. The expression of EMT-related markers was detected by Western blot.we transfected uPA-overexpressing and control HSAEpiCs cells with the uPAR shRNA silencing vector, pSuper-shuPAR. The expression of EMT-related markers were detected by Western blot.Results1. Tests on human lung tissue sections(1) The expression of EMT in patient small airway epitheliumWe observed significant immunostaining of vimentin in small airway epithelial cells from a patient with COPD, while E-cadherin expression was decreased. A marked increase in the number of vimentin positive cells was observed within the small airway epithelium with COPD compared with controls.(2) uPA and uPA R expression in human small airway epitheliumuPA and uPAR expression were increased in the epithelium of distal airways from smokers and patients with COPD, compared with non-smokers. We observed a significant increase in uPA and uPAR levels in the epithelium of patients with COPD compared with non-smokers and smokers without COPD.(3) Correlation analysis between the expression of EMT、uPA、uPAR and lung functionWe observed a significant inverse correlation between FEV1%and vimentin expression (r=-0.461, P<0.01), uPA expression (r=-0.435, P<0.01) and uPAR expression (r=-0.564, P<0.01). uPA expression in small airways was correlated with the number of vimentin positive cells in the small airways (r=0.655, P<0.01). A significant correlation between the mean staining density of uPAR and the number of vimentin positive cells in the small airway epithelium was also observed (r=0.701, P＜0.01).2. Experiments on cell line in vitro(1) CSE-induced EMT in cultured HSAEpiCsAfter72h with5%CSE, HSAEpiCs cells, which typically appear epithelial with well-developed cell junctions, acquired a spindle shape and generally losed cell contacts.The expression of E-cadherin and a-catenin epithelial markers were significantly decreased in HSAEpiCs at both mRNA and protein levels in response to5%CSE. In contrast, CSE increased the expression of N-cadherin and a-SMA in HSAEpiCs.Exposure of HSAEpiCs cells to CSE promotes cell migration.(2) uPAR signaling pathway is required for CSE-induced EMT in HSAEpiCsExpression of uPAR mRNA and protein were markedly increased following CSE exposure.Cells with5%CSE for72h revealed that uPAR knockdown caused a decrease in the population of spindle shaped cells. CSE-treated control vector HSAEpiCs exhibited loss of cell surface E-cadherin and a-catenin, whereas N-cadherin and a-SMA were clearly detected, in keeping with the changes observed with parental cells. In contrast, CSE-treated shuPAR cells exhibited increased E-cadherin and a-catenin and decreased N-cadherin and a-SMA expression.We observed a significant increase in phosphorylated Akt in CSE-treated HSAEpiCs. Western blot analysis revealed activation of PI3K-dependent Akt phosphorylation in parental cells and cells transduced with empty vector following treatment with CSE. In contrast, the level of phosphorylated Akt was substantially inhibited in shuPAR cells cultured with CSE compared. Treatment of cells with LY294002blocked the induction of GSK-3β phosphorylation, Snail and a-SMA. LY294002also preserved expression of the epithelial cell marker E-cadherin in HSAEpiCs treated with CSE.(3) uPA is required for CSE-induced EMT in HSAEpiCs cellsHSAEpiCs cells increased mRNA and protein expression of uPA in response to stimulation with CSE.The amiloride preserved epithelial cell morphology and cell contacts in HSAEpiCs cells that were exposed to CSE. The amiloride inhibited the increase in cell migration observed in HSAEpiCs cells treated with CSE. Cells in the presence of both amiloride and CSE increased levels of E-cadherin and a-catenin’decreased levels of N-cadherin and vimentin expression compared with control cells treated with CSE.Phase-contrast imaging of uPA-overexpressing HSAEpiCs cells revealed loss of cell contacts and acquired a spindle shape. Transient transfection with uPA increased cell migration compared with control cells. uPA overexpression cells induced expression of vimentin and decreased E-cadherin expression.pSuper-shuPAR partially decreased vimentin protein expression and increased E-cadherin protein expression compared with control uPA-overexpressing cells, suggesting that uPAR gene silencing may partly reverse uPA mediated EMT. Conclusionwe demonstrate for the first time that uPA and uPAR are involved in the EMT process in the small airways of patients with COPD.