Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1α in mesenchymal transition.

Irene M J Eurlings,Niki L Reynaert,Fien M Verhamme,Twan Van Den Beucken,Mieke A Dentener,Harry R Gosker,Ken R Bracke,Emiel F M Wouters,C C De Theije,Oliver Eickelberg

doi:10.1371/journal.pone.0107757

Irene M J Eurlings, Niki L Reynaert + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0107757

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Journal: PloS one	Publication Date: Oct 16, 2014
Citations: 64	License type: CC BY 4.0

Affiliation: Maastricht University, Ghent University Hospital

Abstract

In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-β and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-β signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process.

Highlights

Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is progressive and not fully reversible
To test the effect of cigarette smoke on these markers we first tested the effect of cigarette smoke extract (CSE) on viability of A549 and BEAS2B cells
In this study it is shown that CSE induced epithelial-mesenchymal transition (EMT) in alveolar and bronchial epithelial cells

Summary

Introduction

Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is progressive and not fully reversible. The airflow limitation is often ascribed to remodeling, which consists of airway wall thickening and/or emphysema [1]. The association between remodeling and cigarette smoking, the main risk factor for COPD, has been established in both human and animal models [2,3]. Most of these studies show airway wall thickening based on image analysis. Studies into the molecular changes in extracellular matrix associated with small airway remodeling in COPD and mechanisms involved are few, and mainly focus on collagen, fibronectin and glycosaminoglycan deposition [4,5]. The bronchial and alveolar compartment are mostly considered independent entities we recently showed that the matrix of small airway walls and alveolar walls of COPD patients displays very similar features of remodelling [10]

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