Rapamycin inhibits transforming growth factor β1-induced fibrogenesis in primary human lung fibroblasts

Abstract

Purpose: The present study was designed to determine whether rapamycin could inhibit transforming growth factor β1 (TGF-β1)-induced fibrogenesis in primary lung fibroblasts, and whether the effect of inhibition would occur through the mammalian target of rapamycin (mTOR) and its downstream p70S6K pathway.

Materials and methods: Primary normal human lung fibroblasts were obtained from histological normal lung tissue of 3 patients with primary spontaneous pneumothorax. Growth arrested, synchronized fibroblasts were treated with TGF-β1 (10 ng/mL) and different concentrations of rapamycin (0.01, 0.1, 1, 10 ng/mL) for 24 h. We assessed m-TOR, p-mTOR, S6K1, p-S6K1 by Western blot analysis, detected type III collagen and fibronectin secreting by ELISA assay, and determined type III collagen and fibronectin mRNA levels by real-time PCR assay.

Results: Rapamycin significantly reduced TGF-β1-induced type III collagen and fibronectin levels, as well as type III collagen and fibronectin mRNA levels. Furthermore, we also found that TGF-β1-induced mTOR and p70S6K phosphorylation were significantly down-regulated by rapamycin. The mTOR/p70S6K pathway was activated through the TGF-β1-mediated fibrogenic response in primary human lung fibroblasts.

Conclusion: These results indicate that rapamycin effectively suppresses TGF-β1-induced type III collagen and fibronectin levels in primary human lung fibroblasts partly through the mTOR/p70S6K pathway. Rapamycin has a potential value in the treatment of pulmonary fibrosis.

Fig. 1

(A) Characterization of primary human lung fibroblasts. All primary human lung fibroblasts cells displayed typical spindle-shaped morphology under a light microscopy (×40). (B) Characterization of primary human lung fibroblasts. Indirect immunofluorescence staining of primary human pulmonary fibroblasts for vimentin, fibronectin, type III collagen, von Willebrand factor (vWF), pro-surfactant protein C (pro-SPC) and α-smooth muscle actin (α-SMA). Original magnification was ×100. (C) Characterization of primary human lung fibroblasts. Indirect immunofluorescence staining of pulmonary artery endothelial cells for vWF, alveolar type II cells for pro-SPC, and pulmonary artery smooth muscle cells for α-SMA. Original magnification was ×100.

Fig. 2

Assessment of cell viability by MTT. Primary human lung fibroblasts were treated with TGF-β1 (10 ng/mL) and various concentrations of rapamycin (0.01, 0.1, 1.0 and 10 ng/mL). No difference was found in viability between cells treated with rapamycin and TGF-β1 for 24 h and untreated cells (p>0.05). There was also no differernce between TGF-β1 group and the untreated group (p>0.05). Data are representatives of 3 independent experiments. TGF-β1, transforming growth factor β1.

Fig. 3

(A) Rapamycin inhibits TGF-β1-induced type III collagen level in the culture medium of human lung fibroblast, evidenced by ELISA. Type III collagen level after 24-h incubation with medium alone (control), 0.01, 0.1, 1.0, and 10 ng/mL rapamycin and 10 ng/mL TGF-β1. No significant difference was found between different concentration groups of rapamycin. (B) Rapamycin inhibits TGF-β1-induced fibronectin level in the culture medium of human lung fibroblast, shown by ELISA. Fibronectin level after 24-h incubation with medium alone (control), 0.01, 0.1, 1.0, and 10 ng/mL rapamycin and 10 ng/mL TGF-β1. Values are mean of three independent experiments. No significant difference was found between different concentration groups of rapamycin. ^*p<0.05 vs. control; ^†p<0.05 vs. TGF-β1 group. TGF-β1, transforming growth factor β1.

Fig. 4

(A) Effects of rapamycin on TGF-β1-induced gene expression of type III collagen in human lung fibroblasts, shown by real-time PCR. Type III collagen mRNA expression after 24-h incubation with medium alone (control), 0.01, 0.1, 1.0, and 10 ng/mL rapamycin and 10 ng/mL TGF-β1. (B) Effects of rapamycin on TGF-β1-induced gene expression of fibronectin in human lung fibroblasts, shown by real-time PCR. Fibronectin mRNA expression after 24-h incubation with medium alone (control), 0.01, 0.1, 1.0, and 10 ng/mL rapamycin and 10 ng/mL TGF-β1. Values are mean of three independent experiments. ^*p<0.05 vs. control; ^†p<0.05 vs. TGF-β1 group; ^‡p<0.05 vs. 0.01 ng/mL rapamycin group. TGF-β1, transforming growth factor β1.

Fig. 5

(A) Effects of rapamycin on TGF-β1-induced mTOR-Ser2448 phosphorylation in human lung fibroblasts, shown by Western blotting. mTOR-Ser2448 phosphorylation after 24-h incubation with medium alone (control), 0.01, 0.1, 1.0, and 10 ng/mL rapamycin and 10 ng/mL TGF-β1. Differences are apparent between the different rapamycin concentration groups (p<0.05). The densities of protein bands were normalized against β-actin and expressed as a ratio. Values are expressed as mean±SEM of three independent experiments. (B) Effects of rapamycin on TGF-β1-induced p70S6K-Thr389 protein production in human lung fibroblasts, examined by Western blotting. p70S6K-Thr389 protein production after 24-h incubation with medium alone (control), 0.01, 0.1, 1.0, and 10 ng/mL rapamycin and 10 ng/mL TGF-β1. The densities of the protein bands were normalized against β-actin and expressed as a ratio. Values are expressed as mean±SEM of three independent experiments. ^*p<0.05 vs. control; ^†p<0.05 vs. TGF-β1 group; ^‡p<0.05 between two groups. TGF-β1, transforming growth factor β1; mTOR, mammalian target of rapamycin.