Submitted on November 15, 2008
Accepted on April 30, 2009
Induction of Epithelial-Mesenchymal Transition in Primary Airway Epithelial Cells from Asthmatic Patients by TGF
1
Tillie-Louise Hackett1*, Stephanie Mary Warner2, Dorota Stefanowicz2, Furquan Shaheen2, Dmitri V. Pechkovsky3, Lynne A Murray4, Rochelle Argentieri4, Anthony Kicic5, Stephen M Stick6, Tony R. Bai7, and Darryl A Knight1
1 James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St Paul's Hospital, Vancouver, Canada; Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada,
2 James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, Vancouver, Canada,
3 James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, Vancouver, Canada; Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada,
4 Department of Immunobiology, Centocor Ltd, Radnor, Pennsylvania, United States,
5 Department of Respiratory Medicine, Princess Margaret Hospital for Children, Hay Street, Perth, Western Australia, 6001, Australia; School of Pediatrics and Child Health, University of Western Austrtalia, Perth, Western Australia, Australia; Telethon Institute for Child Health Research, Subiaco, Western Australia, Australia,
6 Respiratory Medicine, Princess Margaret Hospital for Children, 13 Watt Street, Swanbourne, Western Australia, 6010, Australia; School of Pediatrics and Child Health, University of Western Austrtalia, Perth, Western Australia, Australia; Telethon Institute for Child Health Research, Subiaco, Western Australia, Australia,
7 James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St Paul's Hospital, Vancouver, Canada
* To whom correspondence should be addressed. 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 asthmatic (n=8) and non-asthmatic normal subjects (n=10). Monolayer and air-liquid interface-AEC (ALI-AEC) cultures were treated with TGF
1 (10ng/ml) for 72h and assayed for mesenchymal and epithelial markers using quantitative PCR, confocal microscopy and immunoblot. The involvement of BMP-7, Smad3, and MAPK-mediated signaling were also evaluated.
Measurements and Main Results: TGF1 induced EMT in AEC monolayers derived from both asthmatic and normal donors. EMT was characterized by changes in cell morphology, increased expression of mesenchymal markers EDA-fibronectin, vimentin, 
-smooth muscle actin, collagen-1 and loss of epithelial markers E-cadherin and Zonular Occludin-1. Inhibition of TGF1-induced signaling with Smad3-inhibiting siRNA or TGF1 neutralizing antibodies prevented and reversed EMT respectively, while 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: TGF1 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 •
EMT •
TGF1 •
Epithelium
