Proteinase-activated receptors (PARs) are newly identified G-protein-coupled receptors that can be activated by serine proteinases such as thrombin, trypsin, mast cell tryptase and neutrophil cathepsin G [
1,
2]. These proteinases cleave the extracellular amino terminal domain of PARs to create a new NH
2 terminal sequence, which functions as a tethered ligand to initiate each receptor-coupled cell signaling. To date, four PARs have been cloned; PAR
1, PAR
3 and PAR
4 are preferentially activated by thrombin, while PAR
2 are selectively activated by trypsin [
2]. In the respiratory system, PAR
1, PAR
2 and PAR
4 are expressed at different levels depending on the tissues or the cell types (epithelium, endothelium, tracheal smooth muscle and blood vessel), and reportedly modulate cytoskeletal structure and further contribute to the progression of various airway and lung disorders including inflammation and fibrosis [
2‐
4]. For example, in systemic sclerosis patients with pulmonary fibrosis or idiopathic pulmonary fibrosis (IPF) patients, concentrations of thrombin and/or cathepsin G in bronchoalveolar lavage fluid are much higher than those in healthy controls [
5,
6]. Therefore, thrombin receptors such as PAR
1 and/or PAR
4 in lung are thought to contribute to the pathogenesis of lung fibrosis. Indeed, Howell et al [
3] demonstrated that bleomycin-induced fibrotic responses such as collagen accumulation and increases in profibrotic mediator levels were attenuated by PAR
1-knockout, suggesting the involvement of PAR
1 signal in the pathogenic mechanisms. However, contribution of another thrombin receptor, PAR
4, has not been examined. In our recent study, PAR
4 (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 PAR
4 stimulation in pathogenetic mechanisms of fibrosis in vitro.
Pulmonary fibrosis is a final common endpoint pathomechanism in various lung diseases including acute respiratory distress syndrome (ARDS) [
8]. The process is characterized by multiple phenomena such as epithelial activation and damage, an excessive extracellular matrix deposition and a substantial increase in the number of fibroblasts/myofibroblasts [
9], transforming growth factor-β (TGF-β), interleukin-4 and tumor necrosis factor-α being known as inducers of such fibrotic responses [
8,
9]. Recently, phenotypic transition of epithelial cell to mesenchymal cell (epithelial-mesenchymal transition; EMT) has received attention as an important mechanism of progressive increase in the number of myofibroblasts in various fibrotic tissues including kidney and lung [
10‐
12]. Typical alveolar epithelia form a cobblestone-like sheet structure that tightly adhering to neighboring cells or various basal substrates, and play an active role in protecting lung from injury and infection [
13]. Under persistent lung pathogenic insults, integrity and characteristics of alveolar epithelium are disturbed and rearranged to induce morphological or physiological alterations, for example, a loss of cell-cell contact, apoptosis and proliferation. Further, parts of epithelial cells are phenotypically changed to different types of cells like mesenchymal cell, i.e., EMT [
9,
12]. During EMT, the epithelial cells lose their characteristic morphology through a various intermediate stages like a loss of epithelial adhesion molecules such as E-cadherin (a specific epithelial marker) and secretion of matrix metalloproteinase (MMP). Finally, cells are converted to a mesenchymal phenotype and acquire myofibloblast morphology characterized by an elongated cell shape and de novo expression of α-smooth muscle actin (α-SMA, a hallmark of myofibroblast). In several different studies, not only growth factor such as TGF-β, epidermal growth factor (EGF) and interleukin-1 but also some drugs (cyclosporine A and angiotensin ll) have been shown to induce EMT of tubular and alveolar epithelial cells, thereby facilitating the progression of renal and lung fibrosis [
8,
10,
14‐
16].
In the present study, we examined whether stimulation of PAR
4 which is known to be involved in the long-scale cellular responses [
17] modulates epithelial morphology through EMT using primary cultured mouse alveolar epithelial cells and a human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Possible mechanisms of the PAR
4's effects were also analyzed with respect to the involvement of EGF receptor (EGFR) signaling, since this receptor is reportedly transactivated by various extracellular stimuli such as G protein-coupled receptors including PARs [
18‐
20].