Gremlin-mediated decrease in bone morphogenetic protein signaling promotes pulmonary fibrosis

Abstract

Rationale: Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF).

Objectives: To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis.

Methods: Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure.

Measurements and main results: Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice.

Conclusions: The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.

Figure 1.

Induction of gremlin expression in asbestos-exposed mouse lungs. (A) Relative mRNA expression levels of gremlin, noggin and chordin in lung tissues exposed to particulate control (titanium dioxide [TiO₂]) or asbestos at 3 and 14 days as analyzed by quantitative real-time reverse transcription–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of a control gene (TATA-binding protein [TBP]), and are expressed relative to control-1 (set to 1). Error bars represent SEM of the samples (n = 5). (B) Paraffin sections from particulate control (TiO₂) and asbestos-treated lungs at 14 days were stained with a gremlin-specific antibody. The control section was treated with goat IgG isotype control. Positive staining is reddish brown. Original magnification = ×400.

Figure 2.

Down-regulation of bone morphogenetic protein (BMP) signaling in asbestos-exposed mouse lungs. Relative mRNA expression levels of BMPs (A) and the BMP target inhibitor of differentiation (Id) genes (B) in lung tissues exposed to particulate control (TiO₂) or asbestos at 14 days, as analyzed by quantitative real-time reverse transcriptase–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of TBP (TATA-binding protein) and are expressed relative to BMP-2 control-1 (A) or Id1 control-1 (B). Error bars represent SEM of the samples (n = 5). (C) Paraffin sections from particulate control (TiO₂) and asbestos-treated lungs at 14 days were stained for phosphorylated Smad (P-Smad) 1/5/8. Positive staining is reddish brown. Original magnification = ×100.

Figure 3.

Expression of gremlin and Id1 in human asbestosis and idiopathic pulmonary fibrosis (IPF) lung biopsies. Total cellular RNA was isolated from control lung tissue (Ctrl) or IPF lung tissue (IPF) from six subjects, and the expressions of gremlin (A) or Id1 and Id2 (B) were analyzed by quantitative real-time reverse transcriptase–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of TBP (TATA-binding protein) and are expressed relative to control-1 (A) or Id1 control-1 (B). Error bars represent SEM of the samples. (C) Paraffin sections from normal adult lung and lungs of patients with IPF or asbestosis were stained for gremlin. Positive staining is reddish brown. Original magnification = ×200.

Figure 4.

Induction of gremlin by asbestos in cultured epithelial cells in vitro. (A) Normal human bronchial epithelial (NHBE) or A549 cells were treated with the indicated concentrations of TiO₂ or asbestos for 20 hours and gremlin expression levels were analyzed by quantitative real-time reverse transcriptase–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of TBP (TATA-binding protein) and expressed relative to untreated control. (B) NHBE cells were transiently transfected with a bone morphogenetic protein (BMP) responsive [Bre]₂-luciferase promoter construct and exposed to asbestos for 24 hours. Luciferase activities were measured and normalized by comparing them with the activities of cotransfected Renilla luciferase activities. The results are expressed as relative luciferase activities. Error bars represent SEM of the samples (n = 3). *P ⩽ 0.05; **P ⩽ 0.01.

Figure 5.

Transforming growth factor (TGF)-β activation is involved in gremlin mRNA induction in normal human bronchial epithelial (NHBE) cells. (A) NHBE cells were transiently transfected with a TGF-β–responsive (CAGA)₁₂-luciferase promoter construct and exposed to asbestos for 24 hours (compare with Figure 4B). The results are expressed as relative luciferase activities. (B) NHBE cells were treated with asbestos (20 μg/cm²) or TGF-β1 (500 pg/ml) in the presence or absence of the type I TGF-β receptor inhibitor, SB431542 (10 μM), for 20 hours. Gremlin expression levels were analyzed by quantitative real-time reverse transcriptase–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of TBP (TATA-binding protein) and are expressed relative to untreated control. Error bars represent SEM of the samples (n = 3).

Figure 6.

Transforming growth factor (TGF)-β activation and target gene expression in asbestos-exposed mouse lungs. Relative mRNA expression levels of TGF-βs (A) and TGF-β target genes, plasminogen activator inhibitor (PAI)-1 and connective tissue growth factor (CTGF) (B), in particulate control (TiO₂) and asbestos-exposed lungs at 14 days analyzed by quantitative real-time reverse transcriptase–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of TBP (TATA-binding protein) and expressed relative to control-1. Error bars represent SEM of the samples (n = 5). (C) Paraffin sections from particulate control (TiO₂) and asbestos-treated lungs at 14 days were stained for P-Smad2. Positive staining is reddish brown. Original magnification = ×400. (D) Equal amounts of lung tissue lysates were analyzed for P-Smad2 and tubulin (control) protein levels by immunoblotting. Lysate of A549 cells treated with TGF-β1 (0.5 ng/ml) for 45 minutes was used as a positive control. Relative P-Smad2 protein levels are indicated.

Figure 7.

Mitogen-activated protein kinase kinase (MEK) inhibitors block gremlin mRNA induction by asbestos in vitro. Normal human bronchial epithelial (NHBE) cells were treated with inhibitors of MEK (20 μM PD98059; 10 μM U0126) in the presence or absence of asbestos (A) or transforming growth factor (TGF)-β1 (B) for 20 hours. Gremlin expression levels were analyzed by quantitative real-time reverse transcriptase–polymerase chain reaction. The mRNA expression levels were normalized to the expression levels of TBP and are expressed relative to untreated control. Error bars represent SEM of the samples (n = 3).

Figure 8.

Bone morphogenetic protein (BMP)-7 treatment reduces fibrosis in asbestos-exposed mouse lungs. (A) Mice were exposed to TiO₂ or asbestos at Day 0. The asbestos-treated animals then received daily injections of BMP-7 or vehicle (phosphate-buffered saline) from Days 7–14. All mice were killed at Day 14 and the lung hydroxyproline contents were analyzed. (B) Bronchoalveolar lavage (BAL) fluid cell counts. Statistical analyses were performed by one-way analysis of variance and Tukey's post test (n = 5).