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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.

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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.

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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.).

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  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

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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.

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Correspondence to Victor J Thannickal.

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

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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

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