In this study, we demonstrate for the first time that uPAR is a regulator involved in the EMT process in the small airways of patients with COPD. Our results demonstrate a significant increase in uPAR expression levels and in the degree of active EMT in the small airway epithelium of patients with COPD compared with non-smokers and smokers with normal lung function. Furthermore, we observed a significant correlation between uPAR expression and EMT in the small airway epithelium of COPD patients. CSE-induced EMT in cultured HSAEpiCs was accompanied by significant induction of uPAR expression, and targeted silencing of uPAR by shRNA inhibited CSE-induced EMT. Taken together, these data suggest that uPAR plays a critical role in CSE-induced EMT. In addition, we demonstrate that the PI3K/AKT signaling pathway is required for uPAR-mediated EMT in HSAEpiCs. Collectively, these results demonstrate that uPAR participates in the EMT process in the small airway epithelium of patients with COPD.
COPD is characterized by airflow limitation that is not completely reversible. Reduced FEV1 characterizes airflow limitation in COPD, and likely occurs because of small airway disease . Hogg and colleagues showed that obstruction of the small airways in COPD is associated with thickening of the airway wall via remodeling processes related to tissue repair . Fibrosis of the small airways is caused by impaired repair following injury to the bronchiolar epithelium . To date, however, the exact mechanisms underlying small airway remodeling in COPD remain poorly understood. EMT is associated with tissue regeneration and fibrosis. EMT refers to a series of phenotypic and molecular changes that occur during various steps of embryonic development, but also in the development of fibrosis and cancer progression. Epithelial cells, via EMTs, are as important precursors of the fibroblasts and myofibroblasts that arise during the course of fibrosis . Although EMT in the airways is implicated in COPD pathogenesis, the mechanisms leading to EMT in the small airways of patients with COPD remain poorly unclear.
Sohal et al. recently observed potential activation of an EMT program in large airways taken from endobronchial biopsies of smokers and patients with COPD . It should be noted, however, that airway remodeling in COPD is mainly located in small conducting airways, and that large and small airways differ in their anatomical and pathophysiological characteristics. Taking this into account, Milara et al. used primary human bronchial epithelial cells (HBECs) from small human bronchi of nonsmokers, smokers and patients with COPD. They showed that the EMT process was present in bronchial epithelial cells of the small bronchi of smokers and patients with COPD and was activated by cigarette smoke in vitro. Although this study demonstrated that cigarette smoke may induce EMT by modulating the TGF-β1 pathway as well as ROS and cAMP levels in primary HBECs, the molecular mechanisms of EMT in the epithelial cells of small airways in COPD patients are still unclear. Previously, Wang et al. identified uPAR expression as an important factor in the progression of COPD by pathway analysis of a signature set of 203 differentially regulated genes . We also showed that uPAR, which can promote EMT in several tumor cell systems [30–32], is highly expressed in the small airway epithelium of patients with COPD compared with controls . However, to date, the role of active EMT in small airways of patients with COPD remains unknown.
We first assessed the expression of EMT biomarkers by immunostaining in small airway epithelial cells from patients with lung resection. We observed significant expression of the epithelial marker, E-cadherin, in the small airway epithelium of non-smokers while vimentin expression was nearly absent. In contrast, E-cadherin was expressed at lower levels in the small airway epithelium of smokers and patients with COPD; however vimentin was clearly observed within the distal airway epithelium. We also observed a marked increase in the number of vimentin positive staining cells within the small airway epithelium of patients with COPD; however no difference was seen in the number of vimentin positive cells between smokers with COPD and nonsmokers with COPD. From the above results, we conclude that cigarette smoke is not the only major factor promoting active EMT, but that other risk factors contribute to airway EMT in COPD. Subsequently, we assessed uPAR expression in small airway epithelial cells in a large patient cohort. uPAR expression was increased in the epithelium of distal airways from smokers and patients with COPD compared with nonsmokers, especially in patients with COPD, in keeping with our previous study . Furthermore, we observed a significant inverse correlation between FEV1% and uPAR or vimentin expression. A significant correlation was also observed between the uPAR expression and vimentin positive cells (per mm of basal membrane) in the small airway epithelium. These results indicate that increased uPAR levels may promote EMT in small airways, thus accounting for the previously observed reduced capacity in COPD patients.
Cigarette smoke is widely used in in vitro studies because of its relevance in the pathogenesis of COPD. We also used this model to investigate mechanisms underlying EMT in COPD. Treatment of HSAEpiCs with CSE led to the acquisition of a fibroblast-like morphology and general loss of cell contacts. This was accompanied by the downregulation of epithelial markers such as E-cadherin and α-catenin, and simultaneous upregulation of mesenchymal markers such as N-cadherin and α-SMA. The levels of uPAR mRNA and protein were markedly increased after exposure of HSAEpiCs to CSE. To test whether uPAR is responsible for CSE-induced EMT, we suppressed uPAR expression using shRNA. This led to inhibition of CSE-induced EMT in HSAEpiCs, indicating that uPAR is required for CSE-induced EMT in HSAEpiC.
Several cell signaling factors have been implicated in this active EMT process. PI3K/Akt is known to promote EMT by regulating the activity of GSK3β, which targets Snail for degradation by suppressing NF-κB-dependent Snail expression [33, 34]. The activities of GSK3β limit the ability of Snail to function as an E-cadherin transcriptional repressor . Other oncogenes, including Rac1, c-Src and Ras have also been implicated in the EMT process [36–38]. Because PI3K/Akt, Rac1, c-Src and Ras are all activated downstream of uPAR , we therefore favor a model in which CSE-induced uPAR expression activates cell signaling via diverse pathways that are complementary in inducing the full spectrum of cellular changes observed in EMT.
To test this model, we studied a uPAR-dependent cell signaling pathway in HSAEpiCs. We showed that PI3K/Akt is activated in HSAEpiCs following treatment with CSE, and this response was blocked by silencing uPAR. Treatment of cells with the PI3K inhibitor, LY294002, also inhibited CSE-induced GSK3β phosphorylation and Snail expression and preserved cell surface E-cadherin. Our finding that uPAR regulates PI3K/Akt activity, and consequently inhibits GSK3β activity, suggests that the uPAR pathway may also contribute to various functions/phenotypes modulated by PI3K/Akt and GSK3β. In our study, the effect of uPAR on EMT via regulation of a PI3K/Akt/GSK3β signaling module is very significant. The link between uPAR and Akt activation by CSE provides one mechanism by which uPAR may ultimately regulate Snail expression and thus promote EMT.