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

Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, Pennathur S, Martinez FJ, Thannickal VJ - Nat. Med. (2009)

Bottom Line: Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O(2) to reactive oxygen species, have increased in number during eukaryotic evolution.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O(2) to reactive oxygen species, have increased in number during eukaryotic evolution. 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 elucidated. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants. The prototypical member of this family, NOX-2 (gp91(phox)), is expressed in phagocytic cells and mediates microbicidal activities. Here we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-beta1 (TGF-beta1) 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 (H(2)O(2)) is required for TGF-beta1-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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NOX4 is expressed in lungs of human subjects with idiopathic pulmonary fibrosis (IPF) and mediates H2O2 production, myofibroblast differentiation, and serum-stimulated proliferation of IPF-derived mesenchymal cells(a) Immunohistochemical staining demonstrating expression of NOX4 in myofibroblastic foci in lungs of a representative human subject with IPF. Length bar = 100 μm. (b–i) Mesenchymal cells isolated from IPF lung tissues (IPF-MCs) by explant tissue culture and analyzed at passage 2–5. (b) IPF-MCs were transfected with nontargeting (control) siRNA or NOX4 siRNA and treated with/without TGF-β1 (2 ng/ml) for 16 h and analyzed for NOX4 protein (inset) and extracellular H2O2 production. (c–f) The effect of siRNA knockdown of NOX4 in IPF-MCs with/without TGF-β1 (2 ng/ml) on the expression of α-SMA mRNA (c) and protein (f); fibronectin mRNA (d) and protein (f); and NOX4 mRNA (e) and protein (f), as determined by real-time PCR (at 24 h) and Western immunoblotting (at 48 h). (g) Control (nontargeting) and NOX4 siRNA transfected IPF-MCs were treated with/without TGF-β1 (2 ng/ml) for 48 h and conditioned culture media was collected and analyzed for acid-soluble collagen using the Sircol assay. (h,i) The effect of siRNA knockdown of NOX4 on proliferation of IPF-MCs treated with/without serum was determined at 24 h by BrdU incorporation assay (h) and at 48 h by assessment of cell numbers using a coulter counter (i). Values represent mean ± S.E.M.; n = 3–5. *P < 0.001 compared to control (without TGF-β1 or serum) and nontargeting siRNA.
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Figure 2: NOX4 is expressed in lungs of human subjects with idiopathic pulmonary fibrosis (IPF) and mediates H2O2 production, myofibroblast differentiation, and serum-stimulated proliferation of IPF-derived mesenchymal cells(a) Immunohistochemical staining demonstrating expression of NOX4 in myofibroblastic foci in lungs of a representative human subject with IPF. Length bar = 100 μm. (b–i) Mesenchymal cells isolated from IPF lung tissues (IPF-MCs) by explant tissue culture and analyzed at passage 2–5. (b) IPF-MCs were transfected with nontargeting (control) siRNA or NOX4 siRNA and treated with/without TGF-β1 (2 ng/ml) for 16 h and analyzed for NOX4 protein (inset) and extracellular H2O2 production. (c–f) The effect of siRNA knockdown of NOX4 in IPF-MCs with/without TGF-β1 (2 ng/ml) on the expression of α-SMA mRNA (c) and protein (f); fibronectin mRNA (d) and protein (f); and NOX4 mRNA (e) and protein (f), as determined by real-time PCR (at 24 h) and Western immunoblotting (at 48 h). (g) Control (nontargeting) and NOX4 siRNA transfected IPF-MCs were treated with/without TGF-β1 (2 ng/ml) for 48 h and conditioned culture media was collected and analyzed for acid-soluble collagen using the Sircol assay. (h,i) The effect of siRNA knockdown of NOX4 on proliferation of IPF-MCs treated with/without serum was determined at 24 h by BrdU incorporation assay (h) and at 48 h by assessment of cell numbers using a coulter counter (i). Values represent mean ± S.E.M.; n = 3–5. *P < 0.001 compared to control (without TGF-β1 or serum) and nontargeting siRNA.

Mentions: To investigate a potential role for NOX4 in a human fibrotic disease, lung tissue sections from IPF were examined. NOX4 is highly expressed in myofibroblastic foci of the remodeled IPF lung, as determined by immunohistochemical (IHC) staining (Fig. 2a). Additionally, lung mesenchymal cells isolated from explants of IPF lung tissue (IPF-MCs) were studied ex vivo. Similar to our findings in hFLMCs, NOX4 was found to be induced and necessary for TGF-β1-stimulated H2O2 production (Fig. 2b); NOX4 was also required for the induction of α-SMA and fibronectin mRNA (Fig. 2c e) and protein expression (Fig. 2f) by TGF-β1. Differentiated myofibroblasts are known to secrete higher amounts of extracellular collagen. We evaluated the role of NOX4 on collagen secretion by IPF-MCs. Constitutive and TGF-β1-induced secretion of soluble collagen by IPF-MCs were inhibited by siRNA-mediated knockdown of NOX4 (Fig. 2g). We examined the role of NOX4 in IPF-MC proliferation in response to serum; siRNA-mediated knockdown of NOX4 inhibited serum-stimulated proliferation of IPF-MCs (Fig. 2h,i). These data support a critical role for NOX4 in myofibroblast differentiation and proliferation of human IPF-MCs.


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

Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, Pennathur S, Martinez FJ, Thannickal VJ - Nat. Med. (2009)

NOX4 is expressed in lungs of human subjects with idiopathic pulmonary fibrosis (IPF) and mediates H2O2 production, myofibroblast differentiation, and serum-stimulated proliferation of IPF-derived mesenchymal cells(a) Immunohistochemical staining demonstrating expression of NOX4 in myofibroblastic foci in lungs of a representative human subject with IPF. Length bar = 100 μm. (b–i) Mesenchymal cells isolated from IPF lung tissues (IPF-MCs) by explant tissue culture and analyzed at passage 2–5. (b) IPF-MCs were transfected with nontargeting (control) siRNA or NOX4 siRNA and treated with/without TGF-β1 (2 ng/ml) for 16 h and analyzed for NOX4 protein (inset) and extracellular H2O2 production. (c–f) The effect of siRNA knockdown of NOX4 in IPF-MCs with/without TGF-β1 (2 ng/ml) on the expression of α-SMA mRNA (c) and protein (f); fibronectin mRNA (d) and protein (f); and NOX4 mRNA (e) and protein (f), as determined by real-time PCR (at 24 h) and Western immunoblotting (at 48 h). (g) Control (nontargeting) and NOX4 siRNA transfected IPF-MCs were treated with/without TGF-β1 (2 ng/ml) for 48 h and conditioned culture media was collected and analyzed for acid-soluble collagen using the Sircol assay. (h,i) The effect of siRNA knockdown of NOX4 on proliferation of IPF-MCs treated with/without serum was determined at 24 h by BrdU incorporation assay (h) and at 48 h by assessment of cell numbers using a coulter counter (i). Values represent mean ± S.E.M.; n = 3–5. *P < 0.001 compared to control (without TGF-β1 or serum) and nontargeting siRNA.
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Figure 2: NOX4 is expressed in lungs of human subjects with idiopathic pulmonary fibrosis (IPF) and mediates H2O2 production, myofibroblast differentiation, and serum-stimulated proliferation of IPF-derived mesenchymal cells(a) Immunohistochemical staining demonstrating expression of NOX4 in myofibroblastic foci in lungs of a representative human subject with IPF. Length bar = 100 μm. (b–i) Mesenchymal cells isolated from IPF lung tissues (IPF-MCs) by explant tissue culture and analyzed at passage 2–5. (b) IPF-MCs were transfected with nontargeting (control) siRNA or NOX4 siRNA and treated with/without TGF-β1 (2 ng/ml) for 16 h and analyzed for NOX4 protein (inset) and extracellular H2O2 production. (c–f) The effect of siRNA knockdown of NOX4 in IPF-MCs with/without TGF-β1 (2 ng/ml) on the expression of α-SMA mRNA (c) and protein (f); fibronectin mRNA (d) and protein (f); and NOX4 mRNA (e) and protein (f), as determined by real-time PCR (at 24 h) and Western immunoblotting (at 48 h). (g) Control (nontargeting) and NOX4 siRNA transfected IPF-MCs were treated with/without TGF-β1 (2 ng/ml) for 48 h and conditioned culture media was collected and analyzed for acid-soluble collagen using the Sircol assay. (h,i) The effect of siRNA knockdown of NOX4 on proliferation of IPF-MCs treated with/without serum was determined at 24 h by BrdU incorporation assay (h) and at 48 h by assessment of cell numbers using a coulter counter (i). Values represent mean ± S.E.M.; n = 3–5. *P < 0.001 compared to control (without TGF-β1 or serum) and nontargeting siRNA.
Mentions: To investigate a potential role for NOX4 in a human fibrotic disease, lung tissue sections from IPF were examined. NOX4 is highly expressed in myofibroblastic foci of the remodeled IPF lung, as determined by immunohistochemical (IHC) staining (Fig. 2a). Additionally, lung mesenchymal cells isolated from explants of IPF lung tissue (IPF-MCs) were studied ex vivo. Similar to our findings in hFLMCs, NOX4 was found to be induced and necessary for TGF-β1-stimulated H2O2 production (Fig. 2b); NOX4 was also required for the induction of α-SMA and fibronectin mRNA (Fig. 2c e) and protein expression (Fig. 2f) by TGF-β1. Differentiated myofibroblasts are known to secrete higher amounts of extracellular collagen. We evaluated the role of NOX4 on collagen secretion by IPF-MCs. Constitutive and TGF-β1-induced secretion of soluble collagen by IPF-MCs were inhibited by siRNA-mediated knockdown of NOX4 (Fig. 2g). We examined the role of NOX4 in IPF-MC proliferation in response to serum; siRNA-mediated knockdown of NOX4 inhibited serum-stimulated proliferation of IPF-MCs (Fig. 2h,i). These data support a critical role for NOX4 in myofibroblast differentiation and proliferation of human IPF-MCs.

Bottom Line: Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O(2) to reactive oxygen species, have increased in number during eukaryotic evolution.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O(2) to reactive oxygen species, have increased in number during eukaryotic evolution. 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 elucidated. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants. The prototypical member of this family, NOX-2 (gp91(phox)), is expressed in phagocytic cells and mediates microbicidal activities. Here we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-beta1 (TGF-beta1) 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 (H(2)O(2)) is required for TGF-beta1-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.

Show MeSH
Related in: MedlinePlus