Bone morphogenetic protein 4 inhibits pulmonary fibrosis by modulating cellular senescence and mitophagy in lung fibroblasts

Abstract

Background: Accumulation of myofibroblasts is critical to fibrogenesis in idiopathic pulmonary fibrosis (IPF). Senescence and insufficient mitophagy in fibroblasts contribute to their differentiation into myofibroblasts, thereby promoting the development of lung fibrosis. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor, is essential for the early stage of lung development; however, the role of BMP4 in modulating lung fibrosis remains unknown.

Methods: The aim of this study was to evaluate the role of BMP4 in lung fibrosis using BMP4-haplodeleted mice, BMP4-overexpressed mice, primary lung fibroblasts and lung samples from patients with IPF.

Results: BMP4 expression was downregulated in IPF lungs and fibroblasts compared to control individuals, negatively correlated with fibrotic genes, and BMP4 decreased with transforming growth factor (TGF)-β1 stimulation in lung fibroblasts in a time- and dose-dependent manner. In mice challenged with bleomycin, BMP4 haploinsufficiency perpetuated activation of lung myofibroblasts and caused accelerated lung function decline, severe fibrosis and mortality. BMP4 overexpression using adeno-associated virus 9 vectors showed preventative and therapeutic efficacy against lung fibrosis. In vitro, BMP4 attenuated TGF-β1-induced fibroblast-to-myofibroblast differentiation and extracellular matrix (ECM) production by reducing impaired mitophagy and cellular senescence in lung fibroblasts. Pink1 silencing by short-hairpin RNA transfection abolished the ability of BMP4 to reverse the TGF-β1-induced myofibroblast differentiation and ECM production, indicating dependence on Pink1-mediated mitophagy. Moreover, the inhibitory effect of BMP4 on fibroblast activation and differentiation was accompanied with an activation of Smad1/5/9 signalling and suppression of TGF-β1-mediated Smad2/3 signalling in vivo and in vitro.

Conclusion: Strategies for enhancing BMP4 signalling may represent an effective treatment for pulmonary fibrosis.

FIGURE 1

Bone morphogenetic protein (BMP)4 is downregulated in lungs and lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients and bleomycin (BLM)-injured mice. a) Representative Western blot results of BMP4, α-smooth muscle actin (SMA), and Col1 protein expressions in lungs of patients with IPF and control subjects (n=30 per group). b) Densitometric analysis of BMP4, α-SMA, and Col1 in the immunoblots using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference. c) Representative Western blot results of BMP4, α-SMA and Col1 protein expressions in isolated lung fibroblasts from patients with IPF and control subjects (n=4 per group). d) Densitometric analysis of BMP4, α-SMA and Col1 in the immunoblots using GAPDH as the internal reference. e) Representative images show BMP4 (red), E-cadherin (green), α-SMA (green) and nuclei (blue) in lung sections of control and subjects with IPF. Right-hand panels display magnified areas from images indicated by dashed boxes. f, g) Western blot analysis of BMP4, α-SMA, fibronectin (FN) and Col1 protein expression in lung tissues of C57BL/6 mice at day 21 post-BLM or -saline administration (n=3∼10 per group). β-actin was used as a loading control. h) Western blot analysis of BMP4 expression in the lung fibroblasts isolated from C57BL/6 mice at day 21 post-BLM or -saline administration (n=4 per group). β-actin was used as a loading control. i) Quantitative real-time PCR analysis of BMP4 mRNA level in the lung fibroblasts isolated from C57BL/6 mice at day 21 post-BLM or -saline administration (n=5 per group). j) Representative images show BMP4 (red), E-cadherin (green), α-SMA (green) and nuclei (blue) in lung sections of C57BL/6 mice at day 21 post-BLM or -saline administration. Right-hand panels display magnified areas from images indicated by dashed boxes. Scale bars=50 µm. Data are presented as mean±sem. DAPI: 4′,6-diamidino-2-phenylindole. **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 2

Bone morphogenetic protein (BMP)4^+/− mice demonstrate higher mortality and more severe fibrosis after bleomycin (BLM)-induced lung injury. a) Percentages of surviving BMP4^+/− and wild type (WT) (BMP4^+/+) mice were plotted over a 21-day period post-BLM (2.0 mg·kg⁻¹) or -saline administration. BMP4^+/++saline n=12, BMP4^+/−+saline n=10, BMP4^+/++BLM n=25, BMP4^+/−+BLM n=37. b) The corresponding body weight of mice in each group was shown over a 21-day observation period. c) Representative axial (top row) and their corresponding coronal (bottom row) images from different groups determined by micro-computed tomography showing radiological features (n=4 per group). Healthy lungs are black, and diseased lungs were increasingly white (elevated density). d) Lung function parameters including respiratory resistance and dynamic compliance (C_dyn) among different groups were compared at day 21 post-BLM or -saline administration (n=5∼6 per group). e) Masson trichrome staining of left lungs from BMP4^+/+ and BMP4^+/− mice at day 21 post-BLM or -saline administration. Images in the lower panels were magnified from the photomicrographs in the upper panels (n=6 per group). f) Hydroxyproline content of lungs from BMP4^+/+ and BMP4^+/− mice after BLM injury (n=5 per group). g) Quantitative real-time PCR analysis of Col1, Col3, fibronectin (FN) and α-smooth muscle actin (SMA) mRNA levels in lung homogenates of BLM-challenged BMP4^+/+ and BMP4^+/− mice. h) Representative Western blot results of α-SMA, Col1, Col3, FN, BMP4 and TGF-β1 and i) expression of phosphorylated and total Smad1/5/9, Smad2 and Smad3 in lung homogenates of BLM-challenged BMP4^+/+ and BMP4^+/− mice (n=6 per group). β-actin was used as a loading control. j) Immunofluorescence analysis of myofibroblasts in lung sections (nucleus, 4′,6-diamidino-2-phenylindole (DAPI); n=3 per group). Representative images of the staining are shown. Scale bars=50 µm. Arrows indicate myofibroblasts with α-SMA- and vimentin-positive staining. Data are presented as mean±sem. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 3

Adeno-associated virus (AAV)9-bone morphogenetic protein (BMP)4 treatment reverses bleomycin (BLM)-induced pulmonary fibrosis. a) Schematic showing the overexpression of BMP4 in the therapeutic BLM mouse lung fibrosis model. Mice were injected intratracheally with AAV9-BMP4 (1.0×10¹⁰ viral genomes in 60 μL saline per mouse) starting at day 10, and lungs were assessed on day 31 after BLM (2.5 mg·kg⁻¹) administration. b) Axial and their corresponding coronal micro-computed tomography (CT) images were acquired after BLM administration. The lower panels show the representative three-dimensional (3D) images drawn out from micro-CT images, based on different tissues with varying density (n=4 per group). c) Lung function parameters including respiratory resistance and dynamic compliance (C_dyn) among different groups were compared 21 days after virus instillation (n=6 per group). d) Masson's trichrome staining of lung sections from AAV9-BMP4- or AAV9-green fluorescent protein (GFP)-treated mice in the therapeutic mouse lung fibrosis model (n=6 per group). Scale bars=1 mm. Images in the lower panels were magnified from the photomicrographs in the upper panels. Scale bars=100 µm. e) Hydroxyproline contents in the lungs of AAV9-BMP4- or AAV9-GFP-treated mice in the therapeutic lung fibrosis model (n=6). f) Quantitative real-time PCR analysis of Col1, Col3, α-smooth muscle actin (SMA), transforming growth factor (TGF)-β1 and BMP4 mRNA expressions in the lungs of AAV9-BMP4- or AAV9-GFP-treated mice in the therapeutic lung fibrosis model (n=5∼8 per group). g) Representative Western blot analysis of α-SMA, Col1, Col3, fibronectin (FN) and BMP4 and k) expression of phosphorylated (p) and total Smad1/5/9, Smad2 and Smad3 in lung homogenates of AAV9-BMP4- or AAV9-GFP-treated mice in the therapeutic lung fibrosis model (n=4 per group). β-actin was used as a loading control. h–j) Immunofluorescence analysis of α-SMA, vimentin, FN and Col1 expressions in lung sections (nucleus, 4′,6-diamidino-2-phenylindole (DAPI); n=3 per group). Representative images of the staining are shown. Scale bars=50 µm. Yellow colouration indicates representative double-positive cells. Data are presented as mean±sem. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 4

Bone morphogenetic protein (BMP)4 inhibits transforming growth factor (TGF)-β1-induced lung fibroblast activation, differentiation and extracellular matrix (ECM) production. a) Real-time quantitative (q)-PCR analysis of BMP4 mRNA expression in human primary lung fibroblasts stimulated with different concentrations of TGF-β1 for 48 h (n=3). b) Real-time qPCR analysis of BMP4 mRNA expression in human primary lung fibroblasts stimulated with TGF-β1 (10 ng·mL⁻¹) at different time points (n=3). c) Western blot analysis of BMP4, Col1, fibronectin (FN) and α-smooth muscle actin (SMA) protein levels in human primary lung fibroblasts stimulated with different concentrations of TGF-β1 for 48 h (n=3). d) Western blot analysis of BMP4, Col1, FN and α-SMA protein levels in human primary lung fibroblasts stimulated with TGF-β1 (10 ng·mL⁻¹) at different time points (n=3). e) Western blot analysis of α-SMA, FN and Col1 protein expressions in total cell lysates of human idiopathic pulmonary fibrosis (IPF) lung fibroblasts treated with TGF-β1 (10 ng·mL⁻¹, 48 h) in the presence of BMP4 (20 μM) or vehicle (n=6). β-actin was used as a loading control. f) Representative images of Col3 immunostaining in human primary lung fibroblasts treated with TGF-β1 (10 ng·mL⁻¹, 48 h) in the presence of BMP4 (20 μM) or vehicle controls (n=3). g) Western blot analysis of α-SMA, Col1, FN and BMP4 and h) expression of phosphorylated and total Smad1/5/9, Smad2 and Smad3 in total cell lysates of primary lung fibroblasts from BMP4^+/+ and BMP4^+/− mice treated with TGF-β1 (10 ng·mL⁻¹, 48 h) (n=4). β-actin was used as a loading control. i) Western blot analysis of α-SMA, FN and Col1 protein expressions in total cell lysates of wild type (WT) mouse primary lung fibroblasts treated with TGF-β1 (10 ng·mL⁻¹, 48 h) and/or BMP4 (20 μM; n=6). β-actin was used as a loading control. j) Western blot analysis of phosphorylated and total expression of Smad1/5/9, Smad2 and Smad3 in total cell lysates of NIH/3T3 fibroblasts treated with TGF-β1 (10 ng·mL⁻¹) and/or BMP4 (20 μM; n=4). β-actin was used as a loading control. k) Immunofluorescent staining of myofibroblasts in mouse primary lung fibroblasts treated with TGF-β1 (10 ng·mL⁻¹, 48 h) and/or BMP4 (20 μM). Representative images of the staining are shown (n=3). Arrows indicate myofibroblasts with α-SMA- and vimentin-positive staining. Nuclear, 4′,6-diamidino-2-phenylindole (DAPI). l, m) Representative images of Col1 and Col3 immunostaining of TGF-β1-differentiated mouse lung fibroblasts treated with BMP4 or vehicle (n=3). Data are presented as mean±sem. Scale bars=100 µm. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 5

Bone morphogenetic protein (BMP)4 inhibits transforming growth factor (TGF)-β1-induced cellular senescence in primary lung fibroblasts. a) Representative images of senescence-associated β-galactosidase (SA-β-gal) staining in primary lung fibroblasts from BMP4^+/+ and BMP4^+/− mice treated with TGF-β1 (10 ng·mL⁻¹) for 48 h (n=4). Scale bars=200 µm. b) Western blot analysis of p53 and p21 protein expressions in total cell lysates of primary lung fibroblasts from BMP4^+/+ and BMP4^+/− mice treated with TGF-β1 (10 ng·mL⁻¹, 48 h) (n=4). β-actin was used as a loading control. c) Western blot analysis of p53 and p21 in total cell lysates of mouse wild-type (WT) primary lung fibroblasts treated with TGF-β1 (10 ng·mL⁻¹, 48 h) and/or BMP4 (20 μM) (n=4). d–g) Quantitative real-time PCR analysis of interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase (MMP)-9 and TGF-β1 mRNA levels in primary lung fibroblasts from BMP4^+/+ and BMP4^+/− mice treated with TGF-β1 (10 ng·mL⁻¹, 48 h) (n=4). Representative microphotographs showing h) intracellular reactive oxygen species (ROS) and i) mitochondrial ROS generation (n=4). Scale bars=100 µm. Data are presented as mean±sem. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 6

Bone morphogenetic protein (BMP)4 promotes mitophagy and restores mitochondrial dynamics in transforming growth factor (TGF)-β1-stimulated lung fibroblasts. a) Representative Western blot results of Beclin-1, p62, LC3B and Pink1 protein expression in total cell lysates of primary lung fibroblasts from BMP4^+/+ and BMP4^+/− mice treated with TGF-β1 (10 ng·mL⁻¹, 48 h) (n=4). β-actin was used as a loading control. b) Representative Western blot results of mitofusin (MFN)1, MFN2, dynamin-related protein (DRP)1 and mitochondrial fission 1 protein (FIS1) expressions in total cell lysates of primary lung fibroblasts from BMP4^+/+ and BMP4^+/− mice treated with TGF-β1 (10 ng·mL⁻¹, 48 h) (n=4). β-actin was used as a loading control. c) Representative Western blot results of MFN1, MFN2, DRP1 and FIS1 expressions in total cell lysates of wild-type (WT) mouse primary lung fibroblasts treated with TGF-β1 (10 ng·mL⁻¹, 48 h) and/or BMP4 (20 μM) (n=4). β-actin was used as a loading control. d) Mitochondrial fission was visualised by MitoTracker Red staining. Scale bars=25 µm. e) Immunofluorescence analysis of LC3B (red) and mitochondria (MitoTracker, green) in primary lung fibroblasts (n=4). Scale bars=25 µm. After Pink1 short-hairpin (sh)RNA (shPink1) or control shRNA (shNC) was transfected into NIH/3T3 fibroblasts, Pink1 protein expression was measured using f) Western blotting and g) immunofluorescence assays (n=4). Scale bars=100 µm. After shPink1 or shNC was transfected into NIH/3T3 fibroblasts for 24 h, cells were treated with TGF-β1 (10 ng·mL⁻¹) and/or BMP4 (20 μM) for 48 h. h) Col1 and α-smooth muscle actin (SMA) protein expressions were examined with Western blot (n=4). i–k) The expression of α-SMA, fibronectin (FN) and Col1 was visualised using confocal microscopy (n=4). Scale bars=100 µm. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

FIGURE 7

Bone morphogenetic protein (BMP)4 attenuates bleomycin (BLM)-induced cellular senescence, impaired mitophagy and oxidative stress in mouse lungs. a, b) Western blot analysis of p53, p21 and p16 in lung homogenates of BLM-challenged BMP4^+/+ and BMP4^+/− mice (n=6 per group). β-actin was used as a loading control. c, d) Real-time quantitative (q)PCR analysis of p53, p21, tumour necrosis factor (TNF)-α and interleukin (IL)-1β mRNA levels in the lungs of BLM-challenged BMP4^+/+ and BMP4^+/− mice (n=5 per group). e) Western blot analysis of Pink1, NADPH oxidase (NOX)4 and superoxide dismutase (SOD)2 in lung homogenates of BLM-challenged BMP4^+/+ and BMP4^+/− mice (n=6 per group). β-actin was used as a loading control. f) Western blot analysis of Pink1 and p21 protein expressions in lung homogenates of adeno-associated virus (AAV)9-BMP4- or AAV9-green fluorescent protein (GFP)-treated mice in the therapeutic lung fibrosis model (n=4 per group). β-actin was used as a loading control. g) Real-time qPCR analysis of p53 and p21 mRNA expressions in lung homogenates of BLM-challenged BMP4^+/+ and BMP4^+/− mice (n=7∼8 per group). h) Immunofluorescence analysis of α-smooth muscle actin (SMA) (green) and p21 (red), as well as α-SMA (green) and p16 (red) expressions in lung sections (nucleus, 4′,6-diamidino-2-phenylindole (DAPI); n=3 per group). Representative images of the staining are shown. Scale bars=50 µm. Data are presented as mean±sem. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 8

Schematic diagram of the study. Bone morphogenetic protein (BMP)4 ameliorates lung fibrosis by antagonising transforming growth factor (TGF)-β1 signalling, which then attenuates cellular senescence and impaired mitophagy of lung fibroblasts, leading to reduced myofibroblast differentiation and extracellular matrix production. BLM: bleomycin; NOX: NADPH oxidase; mtROS: mitochondrial reactive oxygen species; SASP: senescence-associated secretory phenotype; IL: interleukin; MMP: matrix metalloproteinase; SA-β-gal: senescence-associated β-galactosidase.