Elsevier

Life Sciences

Volume 82, Issues 3–4, 16 January 2008, Pages 210-217
Life Sciences

Pirfenidone inhibits the expression of HSP47 in TGF-β1-stimulated human lung fibroblasts

https://doi.org/10.1016/j.lfs.2007.11.003Get rights and content

Abstract

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-β1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-β1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-β1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disorder characterized by patchy fibrotic areas with fibroblast proliferation and extracellular matrix (ECM) remodeling that results in irreversible distortion of the lung architecture (Selman et al., 2001). The underlying molecular mechanisms by which excessive collagen is deposited in the fibrotic lesions are not fully understood. Recently there is a growing body of evidence suggesting that IPF is a model of abnormal wound healing in response to alveolar epithelial injury/activation associated with the formation of patchy areas of fibroblast/myofibroblast foci, which in turn evolve into fibrosis (Selman et al., 2001). Currently available therapeutic measures for treatment of IPF are ineffective, probably because they primarily base in the concept that inflammation leads to injury and fibrosis in IPF (Selman et al., 2001). Several new anti-fibrotic agents which prevent directly the fibroproliferative response and alveolar epithelial damage are under development for IPF treatment.

Pirfenidone is a novel anti-fibrotic agent that inhibits the progression of fibrosis in experimental models of lung, kidney, and hepatic fibrosis (Di Sario et al., 2004, Hewitson et al., 2001, Iyer et al., 1998, Iyer et al., 1999, Iyer et al., 2000, Kakugawa et al., 2004, Kehrer and Margolin, 1997). While this drug has well-established anti-inflammatory properties including regulation of key growth factors and cytokines (Gurujeyalakshmi et al., 1999, Iyer et al., 2000, Nakazato et al., 2002, Oku et al., 2002), recent clinical trials revealed that it also exhibits therapeutic effects in patients with IPF (Azuma et al., 2005, Raghu et al., 1999). However, the exact mechanisms by which this new compound offers protection against lung fibrosis remain unclear. We found previously using an in vivo, mouse model of bleomycin-induced pulmonary fibrosis that pirfenidone reduced the number of myofibroblasts, type II pneumocytes and interstitial spindle-shaped cells expressing heat shock protein (HSP) 47 in fibrotic lesions (Kakugawa et al., 2004). There was also a marked reduction of fibrotic lesions in this model treated by pirfenidone, as a result of reducing the accumulation and deposition of ECM components represented by collagen within the alveolar septa (Kakugawa et al., 2004). In vitro studies with human leiomyoma cells (Lee et al., 1998) and rat hepatic stellate cells (Di Sario et al., 2002) suggested that the anti-fibrotic effects of pirfenidone might be a direct consequence of reduced mitogenesis and collagen synthesis. However, few studies into the specific effects of pirfenidone on lung fibroblasts in vitro have been reported.

HSP47 is a collagen-binding, stress-inducible protein localized in the endoplasmic reticulum that participates in the intracellular processing, folding, assembly, and secretion of procollagens (Ishida et al., 2006, Saga et al., 1987, Sauk et al., 1994). HSP47 is never released into the extracellular matrix, but irrespective of the tissue site and organ, induction of HSP47 expression is always noted during the process of fibrosis, particularly in and around fibrotic lesions (Abe et al., 2000, Shioshita et al., 2000). HSP47-positive cells, especially myofibroblasts, are now thought to be the main source of collagen synthesis, so these cells therefore proposed to play a central role in the synthesis, deposition, and remodelling of the ECM in pulmonary fibrosis in both human patients and animal models (Ishii et al., 2003, Kakugawa et al., 2004, Kakugawa et al., 2005).

Transforming growth factor (TGF)-β is a profibrotic cytokine crucial in the development of pulmonary fibrosis (Bartram and Speer, 2004). Elevated levels of TGF-β in the lungs have been demonstrated in animal models of lung fibrosis and in humans with IPF (Broekelmann et al., 1991, Khalil et al., 1991), and overexpression of TGF-β in the lung causes severe and irreversible pulmonary fibrosis (Sime et al., 1997). TGF-β also enhances expression of HSP47 and collagen-1 in human lung fibroblasts (Yoshioka et al., 2007). In this study, we investigated the precise in vitro action of pirfenidone on the TGF-β1-induced expression of HSP47 and collagen in the lung using a cultured normal human lung fibroblast cell line (NHLF).

Section snippets

Cells and reagents

Normal human adult lung fibroblasts (Clonetics normal human lung fibroblasts [NHLF]) were purchased from Cambrex (Walkersville, MD). Cells were grown in fibroblast basal medium (FBM; Clonetics-BioWhittaker) supplemented with 2% foetal bovine serum, human recombinant fibroblast growth factor (1.0 μg/ml), insulin (5 mg/ml), gentamicin, and amphotericin-B at 37 °C in a 5% CO2-humidified atmosphere. All experiments were performed after 3–5 cell passages. Pirfenidone was provided by Shionogi & Co.

Effects of pirfenidone on expression of TGF-β1-induced HSP47 and collagen type I mRNA

We first examined the effect of pirfenidone on NHLF proliferation using the Alamar Blue reduction assay. Treatment with pirfenidone tended to decrease cell proliferation of NHLF, although the change was not significant (Fig. 1). The effect of pirfenidone on basal and TGF-β1-induced expression of HSP47 and collagen type I mRNA in NHLF was analysed by northern blotting (Fig. 2, Fig. 3). Treatment with 5 ng/ml of TGF-β1 induced a 4-fold increase in HSP47 mRNA expression in 24 h. Pirfenidone alone

Discussion

The major finding of this study was that pirfenidone directly reduced the enhanced production of both HSP47 and collagen type I mRNA and protein induced by TGF-β1 in human lung fibroblasts in a dose-dependent manner.

In animal models of pulmonary fibrosis induced by bleomycin (Iyer et al., 1998, Iyer et al., 1999, Iyer et al., 2000) and cyclophosphamide (Kehrer and Margolin, 1997), pirfenidone demonstrated both anti-inflammatory and anti-fibrotic activities. In these in vivo studies, treatment

Acknowledgements

The authors thank A. Yokoyama (Nagasaki University School of Medicine) for the excellent technical assistance. This study was supported in part by a research grant from the Ministry of Education, Science, Sports and Culture of Japan.

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