501 Activation of PAR-2 Induces Myofibroblast Transformation via a TGF-β and GSK-3β/β-catenin Dependent Pathway in Tissue Remodeling in the Asthmatic Lung

BACKGROUND: Asthma is a chronic inflammatory lung disease, and airway remodeling denotes the pathophysiologic modifications of normal airway wall structure, including changes in the composition and organization of the airway wall's cellular and molecular constituents. These structural alterations are largely irreversible in chronic severe asthma and lead to symptoms associated with chronic airflow limitation. However, the pathogenetic mechanisms leading to these responses remain unclear. According to recent reports, lung-resident fibroblasts and smooth muscle cells have been implicated in the pathogenesis of airway remodeling. Myofibroblasts are proposed to be the primary effector cells of lung fibrotic responses and are characterized by expression of α-smooth muscle actin (α-SMA) stress fibers. Transforming growth factor (TGF)-β is known to induce the transformation of fibroblasts to myofibroblasts. Protease activated receptor (PAR)-2, a G-protein-coupled receptor activated by serine proteases such as trypsin and mast cell tryptase has been recognized as a key molecule in inflammation and fibrotic changes. We hypothesized that activation of PAR-2 induces TGF-β and α-SMA expression and hence may be one of the potential mechanisms of airway remodeling in asthma. METHODS: Cultured human lung fibroblasts (MRC5) were exposed to trypsin (5 nM) or a specific activating peptide, PAR-2AP. Secreted TGF-β was measured using ELISA. Cell associated α-SMA was assessed by Western blot analysis and immunostaining and activation of downstream signaling pathways was assessed by Western analysis. RESULTS: Activation of PAR-2 by trypsin or PAR-2AP induced TGF-β secretion that peaked between 4 and 8 hours. These were correlated with activations of c-fos and c-jun. Induction of α-SMA expression peaked between 4 and 24 hours. Treatment with trypsin or PAR-2AP also induced phosphorylation of GSK-3β on serine 9 and nuclear translocation of β-catenin. CONCLUSIONS: Activation of PAR-2 induces TGF-β secretion through the AP-1 transcription factor complex leading to myofibroblast transformation via the GSK-3β/β-Catenin Pathway.