Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Mar 15;177(6):638-45.
doi: 10.1164/rccm.200708-1291OC. Epub 2007 Dec 20.

An essential role for fibronectin extra type III domain A in pulmonary fibrosis

Andrés F Muro  1 , Federico A Moretti, Bethany B Moore, Mei Yan, Rachelle G Atrasz, Carol A Wilke, Kevin R Flaherty, Fernando J Martinez, Jessica L Tsui, Dean Sheppard, Francisco E Baralle, Galen B Toews, Eric S White
Affiliations

An essential role for fibronectin extra type III domain A in pulmonary fibrosis

Andrés F Muro et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Tissue fibrosis is considered a dysregulated wound-healing response. Fibronectin containing extra type III domain A (EDA) is implicated in the regulation of wound healing. EDA-containing fibronectin is deposited during wound repair, and its presence precedes that of collagen.

Objectives: To investigate the role of EDA-containing fibronectin in lung fibrogenesis.

Methods: Primary lung fibroblasts from patients with idiopathic pulmonary fibrosis or from patients undergoing resection for lung cancer were assessed for EDA-containing fibronectin and alpha-smooth muscle actin (alpha-SMA) expression. Mice lacking the EDA domain of fibronectin and their wild-type littermates were challenged with the bleomycin model of lung fibrosis. Primary lung fibroblasts from these mice were assayed in vitro to determine the contribution of EDA-containing fibronectin to fibroblast phenotypes.

Measurements and main results: Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more EDA-containing fibronectin and alpha-SMA than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wild-type controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer alpha-SMA-expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-beta and decreased lung fibroblast responsiveness to active TGF-beta in vitro.

Conclusions: The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-beta activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.

PubMed Disclaimer

Figures

<b>Figure 1.</b>
Figure 1.
Extra type III domain A (EDA) cellular fibronectin (cFN) regulation in fibroblasts derived from patients with usual interstitial pneumonia (UIP). (A) EDA cFN and α-smooth muscle actin (α-SMA) expression are markedly increased by Western blot in lung fibroblasts from patients with idiopathic pulmonary fibrosis compared with control lung fibroblasts. The membrane was stripped and reprobed for β-tubulin as a loading control. The blot is representative of two similar experiments of separately derived lysates from each patient. (B) Relative expression of EDA-containing mRNA to total fibronectin transcripts in UIP (n = 8) and normal (n = 8) lung fibroblasts as assessed by quantitative reverse transcriptase–polymerase chain reaction. Data are presented as relative EDA inclusion into fibronectin normalized to total fibronectin expression. *P < 0.05.
<b>Figure 2.</b>
Figure 2.
Extra type III domain A–null (EDA−/−) mice fail to develop significant lung fibrosis after bleomycin-induced lung injury. (A) Lung tissue from EDA−/− and wild-type (WT) controls 21 days after bleomycin injury was assessed for total collagen content by hydroxyproline assay. Data are representative of four independently performed experiments. *P < 0.005. (B) Lung sections of WT (top panels) and EDA−/− (bottom panels) mice 21 d after intratracheal bleomycin stained with trichrome to evaluate collagen deposition and anti–α-smooth muscle actin (α-SMA) to identify myofibroblasts. WT mice displayed nests of α-SMA–positive cells corresponding to areas of collagen deposition (blue on trichrome stain), whereas EDA−/− mice demonstrate minimal α-SMA or collagen staining. In EDA−/− mice, α-SMA expression could be observed in airway (arrowhead) and vascular (arrow) smooth muscle cells. Three mice in each group were examined, with similar findings. PBS = phosphate-buffered saline.
<b>Figure 3.</b>
Figure 3.
Persistent inflammation in the lungs of extra type III domain A–null (EDA−/−) mice after intratracheal bleomycin challenge. (A) Bronchoalveolar lavage (BAL) fluid was collected on the indicated days and total cells counted. Results are expressed as mean number of cells (×104). A minimum of seven mice were tested in each group at each time point. *P = 0.009. Open circles represent wild-type (WT) mice, solid triangles represent EDA−/− mice. (B) Differential cell counting in BAL fluid isolated as in (A). Results are expressed as percentage of total cellular infiltrate. PMN = polymorphonuclear leukocytes. Open circles represent WT mice, solid triangles represent EDA−/− mice. EDA cellular fibronectin is dispensable for transforming growth factor (TGF)-β1 production, but necessary for activation. (C) Twenty-four-hour conditioned media from WT and EDA−/− lung fibroblasts were assayed for TGF-β1 by ELISA. No difference in 24-hour TGF-β1 production was observed. (D) Activation of TGF-β is impaired in EDA−/− fibroblasts. WT and EDA−/− fibroblasts were cocultured overnight with mink lung epithelial cells expressing a portion of the plasminogen activator inhibitor-1 promoter fused to luciferase. Reporter constructs cultured alone were a negative control. EDA−/− cells possessed significantly less TGF-β activity than WT cells. *P = 0.0023.
<b>Figure 4.</b>
Figure 4.
Increased mortality in extra type III domain A–null (EDA−/−) mice after high-dose intratracheal bleomycin. By 29 days, 69.9% of wild-type (WT) mice but only 12.7% of EDA−/− mice had survived (P < 0.01 by the log-rank test). Data are pooled from three experiments encompassing 28 EDA−/− mice and 22 WT mice.
<b>Figure 5.</b>
Figure 5.
Extra type III domain A (EDA) cellular fibronectin (cFN) is dispensable for early transforming growth factor (TGF)-β1 signaling. Lung fibroblasts from EDA−/− and wild-type (WT) mice were serum starved, treated without (SF) or with TGF-β1 (2 ng/ml) for the indicated time points, and assayed for phosphorylated Smad2 (pSmad2) and total Smad2 by Western blot. EDA−/− and WT fibroblasts equally demonstrate robust Smad2 phosphorylation by 1 hour after TGF-β1 treatment.
<b>Figure 6.</b>
Figure 6.
Extra type III domain A (EDA)–containing fibronectin rescues EDA−/− cell phenotypic deficiencies in transforming growth factor (TGF)-β1 responsiveness and activation. (A) Serum-starved EDA−/− lung fibroblasts were cultured in the absence (−) or presence (+) of TGF-β1 (2 ng/ml) on tissue culture plastic (TCP) or EDA-containing cellular fibronectin (EDA cFN) for 24 hours. Cell lysates and RNA were collected for (A) α-smooth muscle actin (α-SMA) or (B) collagen I by Western blot (top panel) and semiquantitative reverse transcriptase–polymerase chain reaction (bottom panel). (C) EDA−/− fibroblasts were cocultured overnight on TCP (−) or EDA-containing cFN (+) with mink lung epithelial cells expressing a portion of the plasminogen activator inhibitor-1 promoter fused to luciferase. The next day, lysates were assessed for luciferase activity. *P = 0.0069.
<b>Figure 6.</b>
Figure 6.
Extra type III domain A (EDA)–containing fibronectin rescues EDA−/− cell phenotypic deficiencies in transforming growth factor (TGF)-β1 responsiveness and activation. (A) Serum-starved EDA−/− lung fibroblasts were cultured in the absence (−) or presence (+) of TGF-β1 (2 ng/ml) on tissue culture plastic (TCP) or EDA-containing cellular fibronectin (EDA cFN) for 24 hours. Cell lysates and RNA were collected for (A) α-smooth muscle actin (α-SMA) or (B) collagen I by Western blot (top panel) and semiquantitative reverse transcriptase–polymerase chain reaction (bottom panel). (C) EDA−/− fibroblasts were cocultured overnight on TCP (−) or EDA-containing cFN (+) with mink lung epithelial cells expressing a portion of the plasminogen activator inhibitor-1 promoter fused to luciferase. The next day, lysates were assessed for luciferase activity. *P = 0.0069.
<b>Figure 6.</b>
Figure 6.
Extra type III domain A (EDA)–containing fibronectin rescues EDA−/− cell phenotypic deficiencies in transforming growth factor (TGF)-β1 responsiveness and activation. (A) Serum-starved EDA−/− lung fibroblasts were cultured in the absence (−) or presence (+) of TGF-β1 (2 ng/ml) on tissue culture plastic (TCP) or EDA-containing cellular fibronectin (EDA cFN) for 24 hours. Cell lysates and RNA were collected for (A) α-smooth muscle actin (α-SMA) or (B) collagen I by Western blot (top panel) and semiquantitative reverse transcriptase–polymerase chain reaction (bottom panel). (C) EDA−/− fibroblasts were cocultured overnight on TCP (−) or EDA-containing cFN (+) with mink lung epithelial cells expressing a portion of the plasminogen activator inhibitor-1 promoter fused to luciferase. The next day, lysates were assessed for luciferase activity. *P = 0.0069.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Matsuyama W, Watanabe M, Shirahama Y, Mitsuyama H, Higashimoto I, Osame M, Arimura K. Discoidin domain receptor 1 contributes to the survival of lung fibroblast in idiopathic pulmonary fibrosis. Am J Pathol 2006;168:866–877. - PMC - PubMed
    1. Gharaee-Kermani M, Gyetko MR, Hu B, Phan SH. New insights into the pathogenesis and treatment of idiopathic pulmonary fibrosis: a potential role for stem cells in the lung parenchyma and implications for therapy. Pharm Res 2007;24:819–841. - PubMed
    1. Hernnas J, Nettelbladt O, Bjermer L, Sarnstrand B, Malmstrom A, Hallgren R. Alveolar accumulation of fibronectin and hyaluronan precedes bleomycin-induced pulmonary fibrosis in the rat. Eur Respir J 1992;5:404–410. - PubMed
    1. Kuhn C III, Boldt J, King TE Jr, Crouch E, Vartio T, McDonald JA. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis 1989;140:1693–1703. - PubMed
    1. Pankov R, Yamada KM. Fibronectin at a glance. J Cell Sci 2002;115:3861–3863. - PubMed

Publication types