Abstract
BackgroundProteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.MethodsEMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).ResultsStimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.ConclusionPAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.
Highlights
Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis
We examined whether stimulation of PAR4 which is known to be involved in the long-scale cellular responses [17] modulates epithelial morphology through epithelialmesenchymal transition (EMT) using primary cultured mouse alveolar epithelial cells and a human lung carcinoma-derived alveolar epithelial cell line (A549 cells)
Morphological changes induced by PAR4 agonists Figure 1 shows the typical morphological images of primary cultured alveolar epithelial cells assessed by phase contrast microscopy
Summary
Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Howell et al [3] demonstrated that bleomycin-induced fibrotic responses such as collagen accumulation and increases in profibrotic mediator levels were attenuated by PAR1-knockout, suggesting the involvement of PAR1 signal in the pathogenic mechanisms Contribution of another thrombin receptor, PAR4, has not been examined. PAR4 (mRNA/protein) has been demonstrated to be highly expressed in primary cultured mouse alveolar epithelial cells [7] This enabled us to test the involvement of PAR4 stimulation in pathogenetic mechanisms of fibrosis in vitro
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