Involvement of ER stress, PI3K/AKT activation, and lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis

Abstract

Pulmonary fibrosis is characterized by fibroblast proliferation and extracellular matrix remodelling, leading to respiratory insufficiency. The mechanisms underlying this progressive and devastating disease remain unclear. Conditions that can impair the function of the endoplasmic reticulum (ER) cause accumulation of unfolded or misfolded proteins, resulting in ER stress and activation of the unfolded protein response (UPR). ER stress has been implicated in many conditions including cancer, diabetes, obesity, and inflammation. It is also involved in lung fibrosis, through myofibroblastic differentiation of fibroblasts; however, the precise role of ER stress in lung fibrosis is unknown. The current study aimed to investigate the underlying mechanisms of ER stress inhibitors in the treatment of bleomycin-induced lung fibrosis. We demonstrated that bleomycin can activate ER stress associated proteins, including GRP78, CHOP, and ATF-4, both in vitro and in vivo. PI3K/AKT acts upstream of ER stress to affect lung fibroblast proliferation, resulting in bleomycin-induced pulmonary fibrosis. Treatment with ER stress inhibitors or a PI3K inhibitor caused a reduction in fibroblast proliferation and improved pulmonary function. The relationship between PI3K/AKT/mTOR and ER stress in pulmonary fibrosis, and the application of PI3K inhibitors and ER stress inhibitors in the treatment of pulmonary fibrosis require further investigation.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Bleomycin-induced pulmonary fibrosis was associated with ER stress activation. Mice with intratracheal administration of bleomycin (2 U/kg) or saline (vehicle) were sacrificed 14 days later and the lung specimens were harvested for (A Left) histological analysis with HE, Picro Sirius red and Masson’s trichrome staining followed by (A Right) quantification, (B) The results of total collagen assay, (C) Western blot analysis (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Figure S1), and (D) immunofluorescence for the expression of proteins associated with ER stress activation.

Figure 2

Bleomycin-induced pulmonary fibrosis was attenuated by treatment with ER stress inhibitors. (A) Flow chart of the experimental procedure. Mice with intratracheal administration of bleomycin (2 U/kg) or saline (vehicle) were treated with or without 4-PBA or TUDCA before (Upper: prevention) or 7 days (Lower: treatment) after bleomycin intratracheal instillation. The mice were sacrificed 14 days later and the lung specimens were harvested for histological analysis with (B) HE, (C) picro pirius red and (D) Masson’s trichrome staining followed by (C and D Right) quantification, (E) The results of total collagen assay and (F) Western blot analysis (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Figure S2) for the expression of proteins associated with ER stress activation.

Figure 3

Bleomycin induced ER stress and AKT activation in murine lung fibroblast culture. (A–D) Cells before or indicated time period after treatment with indicated concentration of bleomycin were subjected to western blot analysis for the expression of proteins associated with (A,B) ER stress or (D) AKT activation, and (C) cell number counting (24 hours). (E–G) Cells were treated without (CTR) or with bleomycin in the absence or presence of ER stress or PI3K inhibitor for 6 hours, followed by western blot analysis for the expression of proteins associated with (E) ER stress or (F) AKT activation. (G) Cells transfected with control, PERK, ATF6 and IRE1 shRNA were treated without or with bleomycin for 6 hours, followed by western blot analysis. (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Figure S3B and C).

Figure 4

Treatment of ER stress inhibitors reduced pulmonary fibrosis and expression of p-AKT and p-mTOR in vivo. Mice with intratracheal administration of bleomycin (2 U/kg) or saline (vehicle) were treated with or without 4-PBA (500 mg/kg, i.p.) or TUDCA (500 mg/kg, i.p.) before (prevention) or 7 days (treatment) after bleomycin intratracheal instillation. The mice were sacrificed 14 days later and the lung specimens were harvested for (A,B,D) immunohistochemical analysis, (C) immunofluorescence, (E) Western blot analysis of lung harvested from Mice before or indicated time periods after intratracheal administration of bleomycin (2 U/kg) and (F) western blot analysis with or without treatment of 4-PBA and TUDC, (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Figure S4).

Figure 5

Bleomycin-induced pulmonary fibrosis was attenuated by treatment with PI3K inhibitor. (A) Flow chart of the experimental procedure. Mice with intratracheal administration of bleomycin (2 U/kg) or saline (vehicle) were treated with or without PI3K inhibitor, LY294002 (LY, 50 mg/kg, i.p.) before (Upper: prevention) or 7 days (Lower: treatment) after bleomycin intratracheal instillation. The mice were sacrificed 14 days later and the lung specimens were harvested for histological analysis with (B) HE, (C) picro pirius red and (D) Masson’s trichrome staining followed by (C and D Right) quantification, (E) the results of total collagen assay, (F and G) immunohistochemical analysis, and (H,I) Western blot analysis for the expression of proteins associated with (H) AKT and (I) ER stress after treatment of PI3K inhibitor, LY294002, in control, prevention and treatment groups. (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Figure S5).

Figure 6

PTEN inhibitor activated the PI3K/AKT pathway in murine lung fibroblast culture and also induced pulmonary fibrosis in vivo. (A,B) Cells treated with or without PTEN inhibitor bpV (pic) for 24 hours were subjected to (A) western blot analysis (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Figure S6), and (B) cell number counting (24 hours). (C–E) Mice with intratracheal administration of bpV (2.5 mm) were sacrificed 1, 3 or 7 days later and the lung specimens were harvested for histological analysis with (C) HE, (D) picro pirius red and (E) Masson’s trichrome staining followed by (D and E Right) quantification, (F) The results of total collagen assay.

Figure 7

Pulmonary function in bleomycin-induced pulmonary fibrosis was improved by treatment with ER stress and PI3K inhibitors. (A,B) Barometric plethysmography was conducted in mice before or 14 days after intratracheal administration of bleomycin (2 U/kg) or saline (vehicle), treated with or without (A) 4-PBA or TUDCA (500 mg/kg, i.p.), or (B) PI3K inhibitor, LY294002 (LY, 50 mg/kg, i.p.) before (prevention) or 7 days (treatment) after bleomycin intratracheal instillation. (C) Barometric plethysmography was conducted in mice before or indicated time periods after intratracheal administration of bpV (2.5 mm).