Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury

Nature Medicine volume 15, pages 1077–1081 (2009)Cite this article

Abstract

Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to reactive oxygen species, have increased in number during eukaryotic evolution1,2. Seven isoforms of the NOX gene family have been identified in mammals; however, specific roles of NOX enzymes in mammalian physiology and pathophysiology have not been fully elucidated3,4. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants5,6. The prototypical member of this family, NOX-2 (gp91phox), is expressed in phagocytic cells and mediates microbicidal activities7,8. Here we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-β1 (TGF-β1) induces NOX-4 expression in lung mesenchymal cells via SMAD-3, a receptor-regulated protein that modulates gene transcription. NOX-4–dependent generation of hydrogen peroxide (H2O2) is required for TGF-β1–induced myofibroblast differentiation, extracellular matrix (ECM) production and contractility. NOX-4 is upregulated in lungs of mice subjected to noninfectious injury and in cases of human idiopathic pulmonary fibrosis (IPF). Genetic or pharmacologic targeting of NOX-4 abrogates fibrogenesis in two murine models of lung injury. These studies support a function for NOX4 in tissue fibrogenesis and provide proof of concept for therapeutic targeting of NOX-4 in recalcitrant fibrotic disorders.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification of NOX-4 as the enzymatic source of extracellular H2O2 production by myofibroblasts and its role in mediating myofibroblast differentiation and contractility.
Figure 2: NOX-4 is expressed in lungs of human subjects IPF, and it mediates H2O2 production, myofibroblast differentiation and serum-stimulated proliferation of IPF-derived mesenchymal cells.
Figure 3: NOX-4 is induced during the fibrogenic phase of bleomycin-induced lung injury in mice, and inhibition of NOX-4 expression activity attenuates pulmonary fibrosis.
Figure 4: RNAi-mediated knockdown of NOX-4 attenuates fibrosis in mice subjected to FITC-induced lung injury.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Kawahara, T., Quinn, M.T. & Lambeth, J.D. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol. Biol. 7, 109 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Bedard, K., Lardy, B. & Krause, K.H. NOX family NADPH oxidases: not just in mammals. Biochimie 89, 1107–1112 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Bedard, K. & Krause, K.H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Geiszt, M. & Leto, T.L. The Nox family of NAD(P)H oxidases: host defense and beyond. J. Biol. Chem. 279, 51715–51718 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Levine, A., Tenhaken, R., Dixon, R. & Lamb, C. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583–593 (1994).

    Article  CAS  PubMed  Google Scholar 

  7. Ahluwalia, J. et al. The large-conductance Ca2+-activated K+ channel is essential for innate immunity. Nature 427, 853–858 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Quie, P.G., White, J.G., Holmes, B. & Good, R.A. In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood. J. Clin. Invest. 46, 668–679 (1967).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tomasek, J.J., Gabbiani, G., Hinz, B., Chaponnier, C. & Brown, R.A. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, 349–363 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Thannickal, V.J., Toews, G.B., White, E.S., Lynch, J.P. III & Martinez, F.J. Mechanisms of pulmonary fibrosis. Annu. Rev. Med. 55, 395–417 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Hinz, B. et al. The myofibroblast: one function, multiple origins. Am. J. Pathol. 170, 1807–1816 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Desmoulière, A., Geinoz, A., Gabbiani, F. & Gabbiani, G. Transforming growth factor-β 1 induces α-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol. 122, 103–111 (1993).

    Article  PubMed  Google Scholar 

  13. Serini, G. et al. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-β1. J. Cell Biol. 142, 873–881 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hinz, B., Mastrangelo, D., Iselin, C.E., Chaponnier, C. & Gabbiani, G. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am. J. Pathol. 159, 1009–1020 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Thannickal, V.J. & Fanburg, B.L. Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor β 1. J. Biol. Chem. 270, 30334–30338 (1995).

    Article  CAS  PubMed  Google Scholar 

  16. Thannickal, V.J. et al. Myofibroblast differentiation by transforming growth factor-β1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J. Biol. Chem. 278, 12384–12389 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Waghray, M. et al. Hydrogen peroxide is a diffusible paracrine signal for the induction of epithelial cell death by activated myofibroblasts. FASEB J. 19, 854–856 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Cucoranu, I. et al. NAD(P)H oxidase 4 mediates transforming growth factor-β1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ. Res. 97, 900–907 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Bonniaud, P. et al. Smad3 null mice develop airspace enlargement and are resistant to TGF-β–mediated pulmonary fibrosis. J. Immunol. 173, 2099–2108 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Zhang, K., Rekhter, M.D., Gordon, D. & Phan, S.H. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study. Am. J. Pathol. 145, 114–125 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Thrall, R.S., Barton, R.W., D'Amato, D.A. & Sulavik, S.B. Differential cellular analysis of bronchoalveolar lavage fluid obtained at various stages during the development of bleomycin-induced pulmonary fibrosis in the rat. Am. Rev. Respir. Dis. 126, 488–492 (1982).

    CAS  PubMed  Google Scholar 

  22. Vittal, R. et al. Modulation of prosurvival signaling in fibroblasts by a protein kinase inhibitor protects against fibrotic tissue injury. Am. J. Pathol. 166, 367–375 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Roberts, S.N. et al. A novel model for human interstitial lung disease: hapten-driven lung fibrosis in rodents. J. Pathol. 176, 309–318 (1995).

    Article  CAS  PubMed  Google Scholar 

  24. Thannickal, V.J. Oxygen in the evolution of complex life and the price we pay. Am. J. Respir. Cell Mol. Biol. 40, 507–510 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Sumimoto, H. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J. 275, 3249–3277 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Lambeth, J.D. Nox enzymes, ROS and chronic disease: an example of antagonistic pleiotropy. Free Radic. Biol. Med. 43, 332–347 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Raghu, G., Weycker, D., Edelsberg, J., Bradford, W.Z. & Oster, G. Incidence and prevalence of idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 174, 810–816 (2006).

    Article  PubMed  Google Scholar 

  28. Horowitz, J.C. et al. Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-β1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor. J. Biol. Chem. 279, 1359–1367 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Hattori, N. et al. Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice. J. Clin. Invest. 106, 1341–1350 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D. Lambeth, Department of Biochemistry, Emory University, for providing the rabbit polyclonal antibody to NOX-4 and A. Jesaitis, Department of Microbiology, Montana State University, for the mouse monoclonal antibody to NOX-2. We thank D. Arenberg, Department of Internal Medicine, University of Michigan, for providing primary lung mesenchymal cells from human subjects with IPF (IPF-MCs). This work was supported by grants from the US National Institutes of Health R01 HL067967 (to V.J.T.) and K08 HL081059 (to J.C.H.) and by a National Institutes of Health–sponsored Lung Tissue Research Consortium grant, N01 HR046162 (to F.J.M.).

Author information

Author notes

  1. Victor J Thannickal

    Present address: Present address: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.,

  2. Louise Hecker and Ragini Vittal: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Louise Hecker, Ragini Vittal, Tamara Jones, Rajesh Jagirdar, Tracy R Luckhardt, Jeffrey C Horowitz, Fernando J Martinez & Victor J Thannickal

  2. Division of Nephrology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Subramaniam Pennathur

Authors
  1. Louise Hecker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ragini Vittal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tamara Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rajesh Jagirdar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tracy R Luckhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeffrey C Horowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Subramaniam Pennathur
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fernando J Martinez
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Victor J Thannickal
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.H. and R.V. designed, conducted and supervised experiments and contributed to manuscript preparation; T.J. and R.J. conducted experiments and contributed to primer design and analysis of gene expression data; T.R.L. contributed to mouse studies; J.C.H. contributed to RNAi studies and protein expression data; S.P. contributed to manuscript preparation; F.J.M. contributed to the studies of humans with IPF; V.J.T. conceived of, designed and supervised the project, and L.H. and V.J.T. wrote the manuscript.

Corresponding author

Correspondence to Victor J Thannickal.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1 and 2 and Supplementary Table 1 (PDF 788 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hecker, L., Vittal, R., Jones, T. et al. NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med 15, 1077–1081 (2009). https://doi.org/10.1038/nm.2005

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2005

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing