Induction of epithelial-mesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-beta1.
BACKGROUND
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.


OBJECTIVE
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)-beta1 (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.


RESULTS
TGF-beta1-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, alpha-smooth muscle actin, and collagen-1, and loss of epithelial markers E-cadherin and zonular occludin-1. Inhibition of TGF-beta1-induced signaling with Smad3-inhibiting siRNA or TGF-beta1-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-beta1 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.
Hackett
Tillie-Louise
TL
James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Disease, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC, Canada. thackett@mrl.ubc.ca
Warner
Stephanie Mary
SM
Stefanowicz
Dorota
D
Shaheen
Furquan
F
Pechkovsky
Dmitri V
DV
Murray
Lynne A
LA
Argentieri
Rochelle
R
Kicic
Anthony
A
Stick
Stephen M
SM
Bai
Tony R
TR
Knight
Darryl A
DA
D000293:Adolescent; D001249:Asthma / Q000209:etiology / Q000378:metabolism / Q000473:pathology*; D016022:Case-Control Studies; D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation / Q000187:drug effects*; D002648:Child; D002675:Child, Preschool; D004847:Epithelial Cells / Q000187:drug effects* / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000502:physiology; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology; D008297:Male; D059630:Mesenchymal Stem Cells; D011994:Recombinant Proteins; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D055815:Young Adult
D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1