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Induction of Epithelial–Mesenchymal Transition in Primary Airway Epithelial Cells from Patients with Asthma by Transforming Growth Factor-β1

¹ James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver, British Columbia, Canada; ² Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada; ³ Department of Immunobiology, Centocor Ltd., Radnor, Pennsylvania; ⁴ Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth, Western Australia, Australia; ⁵ School of Pediatrics and Child Health, University of Western Australia, Nedlands, Western Australia, Australia; and ⁶ Telethon Institute for Child Health Research, Subiaco, Western Australia, Australia

Correspondence and requests for reprints should be addressed to T.-L. Hackett, Ph.D., James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Disease, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6. E-mail: thackett{at}mrl.ubc.ca

Rationale: Airway remodeling in asthma is associated with the accumulation of fibroblasts, the primary cell responsible for synthesis and secretion of extracellular matrix proteins. The process by which the number of fibroblasts increases in asthma is poorly understood, but epithelial–mesenchymal transition (EMT) may play a significant role.

Objectives: To evaluate whether EMT occurs in primary airway epithelial cells (AECs), the mechanisms involved, and if this process is altered in asthmatic AECs.

Methods: AECs were obtained from subjects with asthma (n = 8) and normal subjects without asthma (n = 10). Monolayer and air–liquid interface-AEC (ALI-AEC) cultures were treated with transforming growth factor (TGF)-β1 (10 ng/ml) for 72 hours and assayed for mesenchymal and epithelial markers using quantitative polymerase chain reaction, confocal microscopy, and immunoblot. The involvement of BMP-7, Smad3, and MAPK-mediated signaling were also evaluated.

Measurements and Main Results: TGF-β1–induced EMT in AEC monolayers derived from subjects with asthma and normal donors. EMT was characterized by changes in cell morphology, increased expression of mesenchymal markers EDA-fibronectin, vimentin, -smooth muscle actin, and collagen-1, and loss of epithelial markers E-cadherin and zonular occludin-1. Inhibition of TGF-β1–induced signaling with Smad3-inhibiting siRNA or TGF-β1–neutralizing antibodies prevented and reversed EMT, respectively, whereas BMP-7 had no effect. In ALI-AEC cultures derived from normal subjects, EMT was confined to basally situated cells, whereas in asthmatic ALI-AEC cultures EMT was widespread throughout the epithelium.

Conclusions: TGF-β1 induces EMT in a Smad3-dependent manner in primary AECs. However, in asthmatic-derived ALI-AEC cultures, the number of cells undergoing EMT is greater. These findings support the hypothesis that epithelial repair in asthmatic airways is dysregulated.

Key Words: asthma • epithelial–mesenchymal transition • transforming growth factor-β1 • epithelium

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject The potential of alveolar epithelial type II cells to undergo epithelial–mesenchymal transition (EMT) has been extensively studied. However, it is not known if stratified airway epithelial cells from patients with and without asthma can undergo EMT. What This Study Adds to the Field Transforming growth factor-β1 induces phenotypic markers of EMT in airway epithelial cells from donors with and without asthma via a Smad3-dependent process.