Objective: To address the possible role of injured airway epithelium in initiating transdifferentiation of sub-epithelial fibroblasts into myofibroblasts and accelerating cell proliferation in sub-epithelial fibroblasts, which may be involved in airway hyperresponsiveness in asthma.
Methods: Human primary cultured sub-epithelial fibroblasts were co-cultured with human bronchial epithelial cells (16HBE) which were treated with lipopolysaccharide (LPS) plus mechanical scratch prior to co-culture. The procedure was also performed in the presence or absence of endothelin (ET) receptor A inhibitor (BQ123), transforming growth factor-beta(1) (TGF-beta(1)) neutralized antibody, respectively or simultaneously, followed by immunostaining, Western blotting and bromodeoxyuridine (BrdU) incorporation respectively to detect alpha-SMA expression and cell proliferation in the co-cultured sub-epithelial fibroblasts. Using the inhibitors specific for mitogen-activated protein kinases (MAPKs) pathways, the role of MAPKs pathways in activating the expression of alpha-SMA was evaluated. In addition, the interaction between matrix metalloproteinases (MMPs) and ET-1 was investigated by cell transfection with anti-ET-1 converting enzyme (anti-ECE) mRNA expression plasmid followed by gelatin zymography analysis.
Results: 16HBE treated with LPS plus mechanical injury induced alpha-SMA expression in sub-epithelial fibroblasts and accelerated BrdU incorporation in the cells. BQ123, TGF-beta(1) neutralized antibody, specific inhibitors for p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) were able to block the induction respectively to a certain extent. Phosphorylated p38 MAPK and ERK1/2 were detected in the sub-epithelial fibroblasts 10 min after being co-cultured with injured 16HBE. Compared to normal control (16HBE transfected with pEGFPN(2)) or those cells transfected with anti-ECE mRNA expression plasmids, ET-1 released from the 16HBE cells transfected with pEGFPN(2) into supernatants were increased significantly after the treatment described as above: 16HBE pre-transfected with pEGFP-N(2) expression plasmid before being treated with mechanical scrape plus LPS stimulation: (15.00 +/- 0.86) pg/ml; 16HBE pre-transfected with anti-ECE expression plasmid before being treated with mechanical scrape plus LPS stimulation: (7.57 +/- 0.94) pg/ml (all P < 0.01). At the same time, the activities of MMP-2 and MMP-9 were enhanced.
Conclusions: Injured airway epithelial cells induced the transdifferentiation of sub-epithelial fibroblasts into myofibroblasts, which may be mediated by ET-1 and TGF-beta(1) through MARKs pathways such as p38 MAPK and ERK 1/2.