The major finding of the present study was that TGF-β1 enhanced HSP47 and collagen type I synthesis in A549 cells at both the mRNA and protein levels and that pirfenidone directly inhibited these effects.
The collagen-binding, stress-inducible protein, HSP47, acts as a collagen-specific molecular chaperone in the intracellular processing of procollagen [21
]. In a murine model of bleomycin-induced pulmonary lung fibrosis, HSP47 mRNA and protein expression predominantly increased in α-SMA-positive myofibroblasts of the lung interstitium, and the relative amounts of HSP47 mRNA in the lung significantly correlated with the hydroxyproline content [12
]. In addition to fibroblasts, type II pneumocytes in this mouse model expressed HSP47, suggesting that type II pneumocytes also contribute to lung fibrosis [12
]. The expression of type I procollagen and HSP47 was also significantly higher in type II pneumocytes identified in surgical biopsy specimens from patients with idiopathic usual interstitial pneumonia (UIP) than in those from patients with collagen vascular disease-associated UIP and idiopathic nonspecific interstitial pneumonia [26
], which have better prognoses than idiopathic UIP [30
]. These findings suggest that in addition to fibroblasts, type II pneumocytes also directly contribute to lung fibrosis thorough collagen and HSP47 synthesis.
Pirfenidone exerts both anti-inflammatory and anti-fibrotic activities in animal models of pulmonary fibrosis induced by bleomycin [9
] and cyclophosphamide [13
]. These studies in vivo
showed that pirfenidone obviously reduced the histological and biochemical signs of lung fibrosis including the hydroxyproline content. Pirfenidone also decreased the levels of pulmonary protein and gene expression of some fibrogenic cytokines such as TGF-β1 [10
] and platelet-derived growth factor (PDGF) [14
]. Our recent study in vitro
showed that pirfenidone directly inhibits collagen type I and HSP47 expression in TGF-β1-stimulated human lung fibroblasts [20
]. Together, these findings indicate the potential of pirfenidone as a novel, broad-spectrum anti-fibrotic agent. In addition, pirfenidone suppresses the increased expression of HSP47 in our mouse model of lung fibrosis [12
], suggesting that this drug could change the fibroblast phenotype. Here, we demonstrated a similar inhibitory effect of pirfenidone on collagen type I and HSP47 in A549 cells at both the protein and mRNA levels in vitro
, confirming that the effect of pirfenidone on collagen and HSP47 in vivo
represented a direct effect on type II pneumocytes. In addition to its anti-inflammatory action, pirfenidone might act as an anti-fibrotic agent for patients with IPF by directly inhibiting HSP47 and collagen production in type II pneumocytes as well as in fibroblasts in the human lung. In this context, our immunocytochemical study of collagen I showed that pirfenidone co-administration significantly reduced the ratio (%) of positive cells at 100
μg/ml and significantly inhibited TGF-β1-enhanced HSP47 and collagen type I mRNA expression at 500
μg/ml. This suggests that pirfenidone acts as an anti-fibrotic agent by directly inhibiting both HSP47 and collagen type I mRNA expression, which results in reduced collagen synthesis in type II pneumocytes. In support of this proposal, HSP47 inhibition by antisense oligodeoxynucleotides obviously suppresses collagen production in 3
T6 cells [23
], experimental proliferative glomerulonephritis [31
] and in peritoneal fibrosis [32
], indicating that HSP47 is a promising target for the treatment of fibrotic diseases including IPF. This study thus identified pirfenidone as the first known agent that can control HSP47 and collagen expression in type II pneumocytes.
Recent evidence from studies of pulmonary fibrosis supports the role of EMT in the development of fibroblastic foci in IPF [2
]. Yao and colleagues [33
] showed that TGF-β1 induces EMT in alveolar epithelial cells from SD rats. Kasai and colleagues also reported that TGF-β1, but not TNF-α or interleukin-1β, induced A549 cells to undergo EMT [3
]. Willis and colleagues also demonstrated that cultures of primary rat alveolar epithelial cells and a rat alveolar epithelial cell line undergo EMT in response to TGF-β1 stimulation [5
]. Kim et al. also reported that the main source of mesenchymal expansion is lung epithelial cells from a mouse model of pulmonary fibrosis in vivo
]. The induction of EMT is characterized by the expression of α-SMA, transformation of myofibroblast morphology, the increased formation of stress fibers by F-actin reorganization, and loss of the epithelial marker E-cadherin. Collagen type I and HSP47 expression is also considered to be one of the useful parameters for recognizing EMT [28
]. These recent findings and our present data demonstrate that EMT develops in alveolar epithelial cells mediated by TGF-β1, suggesting that such development represents an important mechanism of myofibroblast production during pulmonary fibrosis. In addition, the present data showed that the over-expression of collagen type I, HSP47 and fibronectin induced by TGF-β1 in A549 cells was inhibited by pirfenidone. TGF-β1-induced loss of E-cadherin in A549 cells was also normalized by pirfenidone while this difference was not significant. In addition, pirfenidone inhibited a mesenchymal morphology induced by TGF-β1. These suggest that pirfenidone might partially inhibit EMT.