Effects of bone morphogenetic protein 4 on TGF-β1-induced cell proliferation, apoptosis, activation and differentiation in mouse lung fibroblasts via ERK/p38 MAPK signaling pathway

Chemicals and reagents

Murine recombinant BMP4 (Cat# 315-27) was purchased from PeproTech (Rocky Hill, NJ, USA), and mouse recombinant TGF-β1 (Cat# CK33) was purchased from novoprotein (SinoBio, Shanghai, China). Anti-proliferating cell nuclear antigen (PCNA, Cat# GB11010) and anti-Survivin (Cat# GB11177) antibodies were purchased from Servicebio (Wuhan, China). Anti-BMP4 antibody (Cat# YT7891) was purchased from Immunoway (TX, USA). Anti-fibroblast activation protein (FAP, Cat# A6349) and anti-Bcl-2 (Cat# A0208) antibodies were purchased from ABclonal Technology (Wuhan, China). Anti-p-ERK (Cat# 4370), Anti-ERK (Cat# 4695), anti-p-p38 MAPK (Cat# 4631), and anti-p38 MAPK (Cat# 8690) antibodies were purchased from Cell Signaling Technology (CA, USA). Anti-α-smooth muscle actin antibody (α-SMA, Cat# A2547) was purchased from Sigma-Aldrich (St. Louis, USA). Anti-β-actin antibody (Cat# ab2118), and the horseradish peroxidase-coupled secondary antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). Other reagents were all purchased from GBCBIO Technologies Inc. (Guangzhou, China) unless otherwise indicated.

Isolation of mouse primary lung fibroblasts

BMP4 heterozygous slie mutant mice (BMP4^+/ −) with C57BL/6J background were provided by Jackson Laboratory (Bar Harbor, Maine, USA). The mice were housed in specific pathogen-free facilities with free access to laboratory chow and water, and the ambient temperature was maintained at 21–24 °C and humidity at 40%–60%. All experimental procedures were approved by the Animal Care and Use Committee of Affiliated First Hospital of Guangzhou Medical University (Acceptance number: 2021433).

PLFs were isolated from the mouse lungs (6∼8 weeks) by combining trypsin digestion and tissue adherent methods, according to previously described methods (Zhao et al., 2016). Briefly, mice were anesthetized by 1.2% Avertin solution. Then, the lung tissue of BMP4^+/ + (Wild-type, WT) or BMP4^+/ − mice at 6-8 weeks was cut into small pieces and washed with PBS until the liquid was clear. Trypsin (2.5 mg/ml) was added to digest the tissue for 15min. Lung tissue was moved into the culture flask, immersed in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA) containing 10% FBS, and placed flasks overnight in a CO₂ incubator at 37 °C. On the second day, the tissue blocks were washed with DMEM for 3 times, and the liquid was sucked away. The tissue block was placed evenly in the bottle. DMEM medium supplemented with 10% FBS was then added into the bottle, and the culture bottle was placed vertically in the cell incubator. After 3 h, gently put the bottle down, put the culture bottle in the incubator.

Edu staining

For 5-ethynyl-2-deoxyuridine Edu assays, the proliferation ability of cells was examined with a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (Cat#: C0075S; Beyotime, Haimen, China). The procedure was performed according to the manufacturer’s instruction. After Edu staining, cells were visualized using a fluorescence microscope (EVOS™ Auto 2, Invitrogen, WA, USA), and the percentage of Edu-positive cells was calculated as the number of Edu-positive cells out of the total number of cells (×100).

Apoptotic cell measurement

WT or BMP4^+/ − PLFs were plated in a 6-well plate and treated with TGF-β1 and/or BMP4 for 48 h. Apoptotic PLFs were measured using Annexin V, fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (R&D Systems, Minneapolis, MN, USA).

Quantitative real-time PCR analysis (qRT-PCR)

Total RNA was extracted from PLFs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) followed by reverse transcription a Color Reverse Transcription Kit (EZBioscience, USA). qRT-PCR was performed using with the above cDNA using qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) according to the manufacturer’s protocol. Amplification was conducted using the following primers: 5′-TTGATACCTGAGACCGGGAAG- 3′ (forward) and 5′-ACATCTGTAGAAGTGTCGCCTC-3′ (reverse) for mouse BMP4, and 5′-GCAATTATTCCCCATGAACG-3′ (forward) and 5′-GGCCTCACTAAACCATCCAA-3′ (reverse) for 18s.

Western blot

Western blot analysis was performed as described previously (Guan et al., 2019). Briefly, whole cell lysates of PLFs were extracted by radioimmune precipitation assay (RIPA) (Beyotime Biotechnology, Jiangsu, China) with protease and phosphatase inhibitors (Roche, USA). After determining the concentration of supernatant using a bicinchoninic acid (BCA) protein assay kit (Biocolor BioScience, Shanghai, China), protein samples of 50 µg per lane were loaded into 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). All the blots were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h, and then incubated with corresponding primary antibodies at 4 °C overnight: anti-BMP4 (1:1,000), anti-β-actin (1:1,000), anti-PCNA (1:1,000), anti-Survivin (1:1,000), anti-Bcl-2 (1:1,000), anti-FAP (1:500), anti-α-SMA (1:1,000), anti-p-ERK (1:1,000), anti-ERK (1:1,000), anti-p-p38 (1:1,000), and anti-p38 (1:1,000). After incubation with corresponding secondary antibodies (1:5,000) with gentle shaking at room temperature for 1 h, the membranes were scanned by Tanon-5200 Imaging System (Tanon). Western blot bands were quantified using Image J by testing the band intensity for each group and normalizing it to β-actin as an internal control.

Data and statistical analysis

Data analysis was performed using GraphPad Prism 6.0 (San Diego, CA, USA), and expressed as means  ± SEM from at least fourth independent experiments. Student’s t-test or one-way analysis of variance (One-way ANOVA) was performed to determine statistical significance. p < 0.05 indicated statistical significance.

BMP4 is decreased in TGF-β1-stimulated mouse primary lung fibroblasts (PLFs)

TGF-β1, an important fibrogenic factor, drives lung fibrosis by inducing fibroblast activation. By stimulation of mouse PLFs with TGF-β1, we found that BMP4 mRNA and protein levels were significantly decreased as demonstrated by qRT-PCR (Fig. 1A) and Western blot (Fig. 1B), indicating that BMP4 deficiency is positively associated with fibroblast activation and lung fibrosis.

BMP4 inhibits TGF-β1-induced cell proliferation in mouse primary lung fibroblasts (PLFs)

Fibroblast/activated fibroblast proliferation occurs at the initial stage of tissue repair in response to injury, and the apoptosis of fibroblasts is critical to restore normal tissue architecture (Lorena et al., 2002). We isolated mouse PLFs from WT or BMP4^+/ − mice, and found BMP4 level was reduced approximately 50.2% in BMP4^+/ − PLFs (Fig. 2A). To investigate whether BMP4 affects the proliferation of fibroblasts/activated fibroblasts, mouse PLFs from WT or BMP4^+/ − were treated with TGF-β1 for 48 h and Edu staining was performed. The results showed that a significant enhancement of fibroblast proliferation was observed in TGF-β1-treated cells, which was then further increased by BMP4 haploinsufficiency (Fig. 2B). In addition, the expression levels of PCNA and Survivin, two critical cell proliferation proteins, were measured by Western blot. As shown in Figs. 2C–2E, treatment with TGF-β1 caused an increased PCNA and Survivin expressions, whereas the TGF-β1-induced upregulation of PCNA and Survivin were also further increased by BMP4 haploinsufficiency. In line with this finding, the TGF-β1-induced upregulation of PCNA and Survivin was significantly hampered by addition of exogenous BMP4 (Figs. 2F–2H). These data suggest that BMP4 can inhibit TGF-β1-induced fibroblast/myofibroblast proliferation.

BMP4 promotes cell apoptosis in TGF-β1-stimulated mouse primary lung fibroblasts (PLFs)

Decreased apoptosis in fibroblasts/myofibroblasts promote the formation of fibrotic lesions in IPF (Fattman, 2008). Flow cytometry was therefore used to investigate whether BMP4 affects apoptosis in PLFs. The results showed that BMP4 haploinsufficiency decreased cellular apoptosis in both unstimulated and TGF-β1-stimulated fibroblasts (Figs. 3A, 3B). Western blot showed that Bcl-2, an anti-apoptotic protein, was also significantly increased by TGF-β1, which was further enhanced by BMP4 haploinsufficiency (Fig. 3C). Likewise, the TGF-β1-induced upregulation of Bcl-2 was also reduced by the introduction of exogenous BMP4 (Fig. 3D). Taken together, these results suggest that BMP4 promotes apoptosis in TGF-β1-stimulated lung fibroblasts.

BMP4 suppresses TGF-β1-induced fibroblast activation and myofibroblast differentiation in mouse primary lung fibroblasts (PLFs)

FAP, a cell surface serine protease, is upregulated on a subset of activated fibroblasts, including fibroblasts in IPF (Kimura et al., 2019). Here, we demonstrated that TGF-β1 induced an upregulation of FAP in PLFs, consistent with fibroblast activation (Fig. 4A). The effect on TGF-β1-induced FAP enhancement was blocked by BMP4 supplement (Fig. 4A), suggesting the inhibitory effects of BMP4 on fibroblast activation. Fibroblasts exposed to TGF-β1 can differentiate into myofibroblasts that oversynthesize collagen and other ECM proteins (Hettiarachchi et al., 2020; Guan et al., 2016a; Guan et al., 2016b). The results showed that BMP4 rescued the TGF-β1-induced upregulation of α-SMA levels (Fig. 4B), indicating the inhibitory effects of BMP4 on myofibroblast differentiation.

BMP4 downregulates TGF-β1-activated ERK1/2/p38 MAPK signaling pathway in primary lung fibroblasts (PLFs)

TGF-β1 can induce fibroblast activation and differentiation via the ERK/p38 MAPK signaling pathway (Nie et al., 2017; Ng et al., 2019). Thus, we examined whether BMP4 mediated TGF-β1-induced fibroblast activation and differentiation via the ERK/p38 MAPK signaling pathway. Western blot assay demonstrated that the p-ERK and p-p38 MAPK were markedly increased in TGF-β1-stimulated mouse PLFs, when compared to control cells. Nevertheless, BMP4 haploinsufficiency significantly enhanced the TGF-β1-induced the phosphorylation of ERK and p38 MAPK (Figs. 5A–5C). Correspondingly, the TGF-β1-induced upregulation of phosphorylated ERK and p38 MAPK were significantly repressed by BMP4 administration (Figs. 5D–5F). These findings indicate that BMP4 effectively blocked TGF-β1-activated ERK/p38 MAPK signaling, which are involved in the attenuation of fibroblast activation and differentiation in lung fibroblasts.