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Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells.

Hisatomi K, Mukae H, Sakamoto N, Ishimatsu Y, Kakugawa T, Hara S, Fujita H, Nakamichi S, Oku H, Urata Y, Kubota H, Nagata K, Kohno S - BMC Pulm Med (2012)

Bottom Line: TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process.We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis.Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.

ABSTRACT

Background: Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

Methods: The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.

Results: TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.

Conclusion: We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

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Immunocytochemistry of fibronectin expression in A549 cells stimulated without (a) or with 5 ng/ml (b, c, d) of TGF-β1 without (b), or with 100 (c) or 500 μg/ml (d) of pirfenidone for 48 h. Original magnification: x400. Pirfenidone decreased fibronectin over-expression induced by TGF-β1. Loss of epithelial marker E-cadherin mRNA in A549 cells caused by TGF-β1 (5 ng/ml) was also normalized by pirfenidone, but the difference did not reach significance (e).
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Figure 5: Immunocytochemistry of fibronectin expression in A549 cells stimulated without (a) or with 5 ng/ml (b, c, d) of TGF-β1 without (b), or with 100 (c) or 500 μg/ml (d) of pirfenidone for 48 h. Original magnification: x400. Pirfenidone decreased fibronectin over-expression induced by TGF-β1. Loss of epithelial marker E-cadherin mRNA in A549 cells caused by TGF-β1 (5 ng/ml) was also normalized by pirfenidone, but the difference did not reach significance (e).

Mentions: Immunocytochemical staining of A549 cells showed that pirfenidone inhibited the TGF-β1-induced over-expression of fibronectin, which is a mesenchymal phenotypic marker (Figure 5a-d). Loss of the epithelial marker E-cadherin mRNA in A549 cells induced by TGF-β1 was also normalized by pirfenidone, but this difference did not reach significance (Figure 5e). In addition to the changes in the phenotypic markers, changes in cell morphology were also assessed under phase contrast light microscopy. Untreated A549 cells show a pebble-like epithelial shape and TGF-β1-treated cells show a spindle-like mesenchymal shape. Pirfenidone partially suppressed TGF-β1-induced mesenchymal morphology (Figure 6).


Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells.

Hisatomi K, Mukae H, Sakamoto N, Ishimatsu Y, Kakugawa T, Hara S, Fujita H, Nakamichi S, Oku H, Urata Y, Kubota H, Nagata K, Kohno S - BMC Pulm Med (2012)

Immunocytochemistry of fibronectin expression in A549 cells stimulated without (a) or with 5 ng/ml (b, c, d) of TGF-β1 without (b), or with 100 (c) or 500 μg/ml (d) of pirfenidone for 48 h. Original magnification: x400. Pirfenidone decreased fibronectin over-expression induced by TGF-β1. Loss of epithelial marker E-cadherin mRNA in A549 cells caused by TGF-β1 (5 ng/ml) was also normalized by pirfenidone, but the difference did not reach significance (e).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3403980&req=5

Figure 5: Immunocytochemistry of fibronectin expression in A549 cells stimulated without (a) or with 5 ng/ml (b, c, d) of TGF-β1 without (b), or with 100 (c) or 500 μg/ml (d) of pirfenidone for 48 h. Original magnification: x400. Pirfenidone decreased fibronectin over-expression induced by TGF-β1. Loss of epithelial marker E-cadherin mRNA in A549 cells caused by TGF-β1 (5 ng/ml) was also normalized by pirfenidone, but the difference did not reach significance (e).
Mentions: Immunocytochemical staining of A549 cells showed that pirfenidone inhibited the TGF-β1-induced over-expression of fibronectin, which is a mesenchymal phenotypic marker (Figure 5a-d). Loss of the epithelial marker E-cadherin mRNA in A549 cells induced by TGF-β1 was also normalized by pirfenidone, but this difference did not reach significance (Figure 5e). In addition to the changes in the phenotypic markers, changes in cell morphology were also assessed under phase contrast light microscopy. Untreated A549 cells show a pebble-like epithelial shape and TGF-β1-treated cells show a spindle-like mesenchymal shape. Pirfenidone partially suppressed TGF-β1-induced mesenchymal morphology (Figure 6).

Bottom Line: TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process.We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis.Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.

ABSTRACT

Background: Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

Methods: The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.

Results: TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.

Conclusion: We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

Show MeSH
Related in: MedlinePlus