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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 findings of collagen type I and HSP47 expression in A549 cells incubated for 48 h with various concentrations of TGF-β1. Collagen expression: 0 (a), 1 (b), 5 (c), and 10 ng/ml (d) of TGF-β1. HSP47 expression: 0 (e), 1 (f), 5 (g), and 10 ng/ml (h) of TGF-β1. Original magnification, x400. Positive rates of collagen type I (i) and HSP47 (j) immunostaining of A549 cells after incubation with TGF-β1 (0, 1, 5, 10 ng/ml) for 48 h. Positive rate of stained cells are significantly increased by TGF-β1 compared with control. Values are means ± SEM of 5 experiments.
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Figure 2: Immunocytochemistry findings of collagen type I and HSP47 expression in A549 cells incubated for 48 h with various concentrations of TGF-β1. Collagen expression: 0 (a), 1 (b), 5 (c), and 10 ng/ml (d) of TGF-β1. HSP47 expression: 0 (e), 1 (f), 5 (g), and 10 ng/ml (h) of TGF-β1. Original magnification, x400. Positive rates of collagen type I (i) and HSP47 (j) immunostaining of A549 cells after incubation with TGF-β1 (0, 1, 5, 10 ng/ml) for 48 h. Positive rate of stained cells are significantly increased by TGF-β1 compared with control. Values are means ± SEM of 5 experiments.

Mentions: We first determined the time course of the TGF-β1 (5 ng/ml) effect on the mRNA expression of collagen type I and HSP47 in A549 cells. The response of collagen type I to 5 ng/ml of TGF-β1 was maximal after 6, 12, 24 and 72 h of incubation (Figure 1a). TGF-β1 at 5 and 10 ng/ml also significantly increased the expression of collagen type I mRNA after 6 h of incubation (Figure 1b). The response of HSP47 to 5 ng/ml TGF-β1 was maximal after 48 and 72 h of incubation (Figure 1c). Figure 1d shows that TGF-β1 at 5 and 10 ng/ml significantly increased HSP47 mRNA expression compared with untreated controls after 48 h. We next examined the effects of TGF-β1 on collagen type I and HSP47 protein expression in A549 cells using immunocytochemical staining. We found that 1, 5 and 10 ng/ml of TGF-β1 increased the number of cells that were immunopositive for both collagen type I and HSP47 at 48 h (Figure 2).


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 findings of collagen type I and HSP47 expression in A549 cells incubated for 48 h with various concentrations of TGF-β1. Collagen expression: 0 (a), 1 (b), 5 (c), and 10 ng/ml (d) of TGF-β1. HSP47 expression: 0 (e), 1 (f), 5 (g), and 10 ng/ml (h) of TGF-β1. Original magnification, x400. Positive rates of collagen type I (i) and HSP47 (j) immunostaining of A549 cells after incubation with TGF-β1 (0, 1, 5, 10 ng/ml) for 48 h. Positive rate of stained cells are significantly increased by TGF-β1 compared with control. Values are means ± SEM of 5 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Immunocytochemistry findings of collagen type I and HSP47 expression in A549 cells incubated for 48 h with various concentrations of TGF-β1. Collagen expression: 0 (a), 1 (b), 5 (c), and 10 ng/ml (d) of TGF-β1. HSP47 expression: 0 (e), 1 (f), 5 (g), and 10 ng/ml (h) of TGF-β1. Original magnification, x400. Positive rates of collagen type I (i) and HSP47 (j) immunostaining of A549 cells after incubation with TGF-β1 (0, 1, 5, 10 ng/ml) for 48 h. Positive rate of stained cells are significantly increased by TGF-β1 compared with control. Values are means ± SEM of 5 experiments.
Mentions: We first determined the time course of the TGF-β1 (5 ng/ml) effect on the mRNA expression of collagen type I and HSP47 in A549 cells. The response of collagen type I to 5 ng/ml of TGF-β1 was maximal after 6, 12, 24 and 72 h of incubation (Figure 1a). TGF-β1 at 5 and 10 ng/ml also significantly increased the expression of collagen type I mRNA after 6 h of incubation (Figure 1b). The response of HSP47 to 5 ng/ml TGF-β1 was maximal after 48 and 72 h of incubation (Figure 1c). Figure 1d shows that TGF-β1 at 5 and 10 ng/ml significantly increased HSP47 mRNA expression compared with untreated controls after 48 h. We next examined the effects of TGF-β1 on collagen type I and HSP47 protein expression in A549 cells using immunocytochemical staining. We found that 1, 5 and 10 ng/ml of TGF-β1 increased the number of cells that were immunopositive for both collagen type I and HSP47 at 48 h (Figure 2).

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