Bone morphogenetic protein-4 inhibitor gremlin is overexpressed in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF), ie, usual interstitial pneumonia in histopathology, is a disease characterized by tissue destruction and active areas of fibroproliferation in the lung. Gremlin (Drm), a member of the cysteine knot family of bone morphogenetic protein (BMP) inhibitors, functions to antagonize BMP-4-mediated signals during lung development. We describe here consistent overexpression of gremlin in the lung interstitium of IPF patients. Quantitative real-time reverse transcriptase-polymerase chain reaction analyses revealed considerably higher levels of gremlin mRNA in lung biopsies from IPF patients, the highest level being 35-fold higher compared to controls. Lung fibroblasts isolated from IPF patients also expressed elevated levels of gremlin, which was associated with impaired responsiveness to endogenous and exogenous BMP-4. Transforming growth factor-beta-induced epithelial-to-mesenchymal transition of A549 lung epithelial cells in culture was also associated with induction of gremlin mRNA expression. In addition, A549 cells transfected to overexpress gremlin were more susceptible to transforming growth factor-beta-induced epithelial-to-mesenchymal transition. Gremlin-mediated inhibition of BMP-4 signaling pathways is likely to enhance the fibrotic response and reduce epithelial regeneration in the lung. The overexpression of this developmental gene in IPF may be a key event in the persistence of myofibroblasts in the lung interstitium and provides a potential target for therapeutic intervention.

Figure 1

Increased secretion of TGF-β activity in lung fibroblasts from IPF patients. A: Fibroblasts isolated from control adult lung (CCL-190) or IPF patient lung (CCL-191 and -134) were cultured on glass coverslips and stained for the myofibroblast marker α-SMA. Red color indicates positive staining. B: Conditioned serum-free medium (24-hour collection) was harvested from fibroblasts, and aliquots of the media from the same number of cells were either analyzed directly for TGF-β activity (active TGF-β) or after heat treatment (total TGF-β). Heat treatment activates latent forms of TGF-β. The results are expressed as relative TGF-β activity (the activity in control medium is set to 1). The error bars represent the SEM of the samples. C: Total cellular RNA was isolated from cultured control (CCL-190) or IPF (CCL-134) fibroblasts and mRNA expression levels of TGF-βs 1 to 3 and CTGF were analyzed by Northern hybridization. The expression of a constant gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used to control loading.

Figure 2

IPF fibroblasts overexpress gremlin. A: Total cellular RNA was isolated from cultured control (CCL-190) or IPF (CCL-191 and -134) fibroblasts, and mRNA expression levels of gremlin and BMP-4 were analyzed by Northern hybridization. The expression of a constant gene, GAPDH, was used to control loading. B: Cell-conditioned medium was collected from the same number of cells and analyzed for gremlin and BMP-4 protein levels by immunoblotting. C: Control lung (CCL-190) and skin (4′-strain) fibroblasts as well as primary fibroblasts isolated locally from patients with IPF (IPF-U.N., see Materials and Methods), and systemic (CRL-1108) or localized (morphea) scleroderma were cultured on glass coverslips and stained for the myofibroblast marker α-SMA. Red color indicates positive staining. D: Total cellular RNA was isolated from control (CCL-190, 4′-strain) and patient (IPF-U.N., CRL-1108, morphea) fibroblasts, and Northern hybridization analyses were performed as in A.

Figure 3

BMP-4 signaling is impaired in IPF fibroblasts. A: Control (CCL-190) or IPF (CCL-191 and -134) fibroblasts were transiently transfected with a BMP-responsive (BRE)₂-luciferase promoter construct (see Materials and Methods). Luciferase activities were measured 48 hours after transfection. The activities were normalized by comparing them with the activities of co-transfected Renilla luciferase. The results are expressed as relative luciferase activities (the activity in control fibroblasts is set to 1). The error bars represent the SEM of the samples. B: Control (CCL-190) or IPF (CCL-191) fibroblasts were transiently transfected, treated with the indicated concentration of BMP-4 for 16 hours, and analyzed for luciferase activity as in A. C: Fibroblasts were treated with the indicated concentrations of BMP-4 for 1 hour, and the levels of phosphorylated Smad1 (P-Smad1) and total Smad1 were analyzed from cell lysates by immunoblotting. Densitometric scanning results normalized to the untreated CCL-190 control are shown below the blots.

Figure 4

Paraffin sections from normal adult and IPF patient lungs were stained for gremlin and BMP-4. Positive staining is reddish-brown. In the normal lungs (two sections) gremlin immunoreactivity was located in alveolar macrophages and cells lining the alveolar wall (arrow). No gremlin immunoreactivity was observed in the parenchyme (P). IPF lungs showed strong immunoreactivity in early parenchymal lesions where the alveolar structure was still preserved (bottom left), whereas in areas of more advanced pulmonary fibrosis staining was intense throughout the parenchyme (second bottom panel, IPF-U.N. cell line was propagated from this patient). In IPF lungs, no gremlin immunoreactivity was observed in the epithelial cells (arrow). BMP-4 immunoreactivity was observed in the normal and IPF lungs in cells lining the alveoli (A, small arrows). The amount of BMP-4-positive cells was similar in both normal and IPF lungs. Control sections were treated with mouse isotype control (not shown) or goat IgG (right). Original magnifications: ×10 or ×40; except the top left panel at ×20.

Figure 5

Increased expression of gremlin in the lungs of IPF patients. A: Total cellular RNA was isolated from control (Ctrl) or IPF patient lung (IPF) tissues from six subjects, and the expression of gremlin (A) or BMP-2, -4, and -7 (B) was analyzed by quantitative real-time RT-PCR. The mRNA expression levels were normalized to the expression of a control gene (actin), and the results are expressed as relative values (gremlin expression in Ctrl-1 is set to 1). The error bars represent SEM of the samples.

Figure 6

TGF-β1 induces gremlin expression in association with EMT in lung epithelial cells. A: A549 lung epithelial cells were cultured on glass coverslips, treated with 1 ng/ml TGF-β1 for 48 hours, and stained for E-cadherin. The staining indicates E-cadherin protein (red) and nuclei (blue). B: A549 cells were treated with TGF-β1 for the indicated times, after which total cellular RNA was isolated and mRNA expression levels of gremlin were analyzed by Northern hybridization. mRNA expression of a constant gene, GAPDH, was used to control loading.

Figure 7

Gremlin overexpression sensitizes A549 cells to TGF-β1-induced EMT. A: Total cellular RNA was isolated from A549 or A549/gremlin cells, and mRNA expression levels of gremlin were analyzed by Northern hybridization. mRNA expression of a constant gene, GAPDH, was used to control loading. B: Cells were transiently transfected with a BMP-responsive (BRE)₂-luciferase promoter construct, and luciferase activities were measured as in Figure 3A. The error bars represent the SEM of the samples. C: A549 and A549/gremlin cells were cultured on glass coverslips, treated with the indicated concentrations of TGF-β1 for 48 hours, and stained for E-cadherin. The staining indicates E-cadherin protein (red) and nuclei (blue).

Figure 8

Potential roles of the TGF-β and BMP-4 signaling pathways in IPF. The expression of TGF-β is already induced during early stages of IPF. TGF-β recruits fibroblasts to the site of injury and induces their activation. TGF-β also stimulates the EMT of alveolar epithelial cells. Pulmonary myofibroblasts persist in IPF and overexpress gremlin, which can inhibit BMP-4-induced regeneration of the epithelium. This may lead to a vicious circle of sustained epithelial injury and TGF-β1 activation. In addition, gremlin blocks BMP-4-mediated myofibroblast apoptosis, which further enhances parenchymal fibrosis. Excessive fibroproliferation leads to the excessive accumulation of the ECM.