Effects of bone morphogenetic protein 4 on TGF- β 1-induced cell proliferation, apoptosis, activation and differentiation in mouse lung fibroblasts via ERK/p38 MAPK signaling pathway

Abstract

Fibroblasts, in particular myofibroblasts, are the critical effector cells in idiopathic pulmonary fibrosis (IPF), a deadly lung disease characterized by abnormal lung remodeling and the formation of "fibroblastic foci". Aberrant activation of TGF-β1 is frequently encountered and promotes fibroblast proliferation, activation, and differentiation in pulmonary fibrosis. Hence, the inhibition of TGF-β1-induced lung fibroblast activation holds promise as a therapeutic strategy for IPF. The present study aimed to investigate the potential effect and underlying mechanisms of bone morphogenetic protein 4 (BMP4) on TGF-β1-induced proliferation, apoptosis, activation and myofibroblast differentiation of adult lung fibroblasts. Here, we demonstrated that BMP4 expression was significantly decreased in TGF-β1-stimulated mouse primary lung fibroblasts (PLFs). BMP4 inhibited proliferation and apoptosis resistance of TGF-β1-stimulated mouse PLFs. BMP4 suppressed TGF-β1-induced fibroblast activation and differentiation in mouse PLFs. We also found that BMP4 inhibited TGF-β1-induced ERK and p38 MAPK phosphorylation. Our findings indicate that BMP4 exerts its anti-fibrotic effects by regulating fibroblast proliferation, apoptosis, activation and differentiation via the inhibition of the ERK/p38 MAPK signaling pathway, and thus has a potential for the treatment of pulmonary fibrosis.

Keywords: Activation; Apoptosis; BMP4; Fibroblasts; Pulmonary fibrosis; TGF-β1.

Figure 1. BMP4 was decreased in TGF- β1-stimulated mouse primary lung fibroblasts (PLFs).

(A) qRT-PCR analysis of BMP4 mRNA levels in mouse PLFs stimulated with TGF- β1 (10 ng/ml) for 48 h. (B) Western blot analysis of BMP4 protein expression in mouse PLFs stimulated with TGF- β1 for 48 h. Data were expressed as mean ± SEM, ^**p < 0.01; ^***p < 0.001.

Figure 2. BMP4 inhibited TGF- β1-induced proliferation of mouse PLFs.

(A–C) Western blot analysis of PCNA and Survivin in total cell lysates of PLFs from BMP4^+/ + and BMP4^+/− mice treated with TGF- β1 (10 ng/ml, 48 h). β-actin was used as a loading control. (D–E) Western blot analysis of PCNA and Survivin protein expressions in total cell lysates of WT PLFs treated with TGF- β1 (10 ng/ml, 48 h) and/or BMP4 (20 µM). β-actin was used as a loading control. Data were expressed as mean ± SEM, ^**p < 0.01; ^****p < 0.0001.

Figure 3. BMP4 promoted cell apoptosis in TGF- β1-stimulated mouse PLFs.

(A) PLFs from BMP4^+/ + and BMP4^+/− mice were treated with TGF- β1 (10 ng/mL) for 48 h. The cells were double-stained with Annexin V-FITC and PI, and then the cellular apoptosis was determined by flow cytometry. (B) The ratio of apoptotic cells was statistically analyzed. (C) Western blot analysis of Bcl-2 in total cell lysates of PLFs from BMP4^+/ + and BMP4^+/− mice treated with TGF- β1 (10 ng/ml, 48 h). β-actin was used as a loading control. (D) Western blot analysis of Bcl-2 protein expression in total cell lysates of WT PLFs treated with TGF- β1 (10 ng/ml, 48 h) and/or BMP4 (20 µM). β-actin was used as a loading control. Data were expressed as mean ± SEM, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 4. BMP4 suppressed TGF- β1-induced fibroblast activation and differentiation in mouse PLFs.

PLFs were incubated with TGF- β1 (10 ng/ml) in the absence or presence of BMP4 (20 µM) for 48 h. Western blot assay was used to detect FAP (A) and α-SMA (B) protein expression. β-actin was used as a loading control. Data were expressed as mean ± SEM, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 5. BMP4 downregulated TGF- β1-activated ERK/p38 MAPK signaling in PLFs.

(A) Western blot analysis of phosphorylated and total ERK or p38 in total cell lysates of PLFs from BMP4^+/ + and BMP4^+/− mice treated with TGF- β1 (10 ng/ml, 48 h). β-actin was used as a loading control. (B) Western blot analysis of phosphorylated and total expression of ERK or p38 in total cell lysates ofPLFs treated with TGF- β1 (10 ng/ml) and/or BMP4 (20 µM). β-actin was used as a loading control. Data were expressed as mean ± SEM, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.