NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury - PubMed (original) (raw)

. 2009 Sep;15(9):1077-81.

doi: 10.1038/nm.2005. Epub 2009 Aug 23.

Affiliations

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

Louise Hecker et al. Nat Med. 2009 Sep.

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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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare no conflict of interest and there are no financial relationships with commercial entities which have interest in the subject of this manuscript.

Figures

Figure 1

Figure 1. Identification of NOX4 as the enzymatic source of extracellular H2O2 production by myofibroblasts and its role in mediating myofibroblast differentiation and contractility

(a) RNA was isolated from human fetal lung mesenchymal cells (hFLMCs) treated with/without TGF-β1 (2 ng/ml) for 18 h and analyzed by Affymetrix (U133A) microarray for members of the NOX/DUOX gene family. Values represent mean ± S.D., n = 3 per group. *P < 0.001 compared to control. ND indicates “not detected” (below threshold). (b) hFLMCs were treated with/without TGF-β1 (2 ng/ml) for the times indicated and cell lysates subjected to SDS-PAGE and Western immunoblotting for NOX4 and GAPDH. (c) Effect of NOX4 siRNA (duplex 4) on extracellular release of H2O2 by hFLMCs treated with/without TGF-β1 (2 ng/ml for 16 h). (d) hFLMCs were pretreated with pharmacologic inhibitors against ALK5 receptor kinase (SB431542; 1 μM), MEK (PD98059; 20 μM), p38 MAPK (SB203580; 6 μM), JNK (SP600125; 100 nM), and then stimulated with TGF-β1 (2 ng/ml × 16 h) prior to measurement of extracellular H2O2 release. (e) Effect of SMAD3 siRNA knockdown on TGF-β1-induced NOX4 expression in hFLMCs, as determined by Western immunoblotting. (f) Effect of siRNA-mediated knockdown of SMAD3 on extracellular H2O2 production stimulated by TGF-β1 (2 ng/ml × 16 h) in hFLMCs. (g) hFLMCs in 3-D collagen matrix were stimulated with/without TGF- β1 (2 ng/ml × 16 h) in the presence/absence of catalase (750 U/ml) and effects on α-smooth muscle actin (α-SMA), fibronectin, and β-actin were determined by Western immunoblotting. (h) Effect of siRNA-mediated silencing of NOX4 in 3D-collagen matrix-embedded hFLMCs on cellular expression of α-SMA, fibronectin, and procollagen-1 treated with/without TGF-β1 (2.5 ng/ml × 72 h), as determined by Western immunoblotting. (i,j) Effect of exogenous catalase (750 U/ml) (i), and siRNA-mediated NOX4 silencing (j) on TGF-β1-induced contractility in 3D-collagen matrices. Values represent mean ± S.E.M.; n = 4. *P < 0.001 compared to controls.

Figure 2

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.

Figure 3

Figure 3. NOX4 is induced during the fibrogenic phase of bleomycin-induced lung injury in mice and inhibition of NOX4 expression/activity attenuates pulmonary fibrosis

(a) C57BL/6 mice were subjected to acute lung injury by airway (intra-tracheal) administration of bleomycin or saline/control on day 0. Following bleomycin injury, mice were euthanized at the indicated time intervals, whole lungs were harvested, and tissue homogenates analyzed by SDS-PAGE and Western immunoblotting for NOX4, NOX2, and β-actin. (b–e) NOX4 siRNA or a nontargeting control siRNA was instilled directly down the trachea of mice at the time of bleomycin injury (day 0), and lungs were analyzed on day 14 or 21. (b) NOX4 expression on day 21 was determined by Western immunoblotting of whole lung homogenates. (c) Fibrosis was assessed on day 14 by H & E staining and Masson’s trichrome blue staining for collagen (top panels); NOX4 and α-SMA expression were assessed by immunohistochemical (IHC) analysis (bottom panels); length bar = 100 μm. (d,e) Whole lung homogenates were analyzed on day 21 for hydroxyproline content (d), and on day 14 for acid-soluble collagen using the Sircol assay (e). Values represent mean± S.E.M.; n = 4–6; *P < 0.01 compared to all other groups. (f) C57BL/6 mice were administered intra-tracheal (IT) bleomycin on day 0. Diphenyleneiodonium (DPI; 1.6 mg/kg) or vehicle control was administered by daily intraperitoneal (IP) injections starting on day 7 for 14 days. Whole lung homogenates were analyzed for acid-soluble collagen using the Sircol assay on day 21. Values represent mean ± S.E.M.; n = 6; *P < 0.05 compared to saline control.

Figure 4

Figure 4. RNAi-mediated knockdown of NOX4 attenuates fibrosis in mice subjected to fluorescein isothiocyanate-induced lung injury

C57BL/6 mice were administered intra-tracheal fluorescein isothiocyanate (FITC) or saline/control on day 0 with nontargeting control siRNA or NOX4 siRNA and lung tissues were analyzed on day 14 or 21. (a) NOX4 expression on day 21 was determined by Western immunoblotting of whole lung homogenates. (b) Fibrosis was assessed by H & E staining and Masson’s trichrome blue staining for collagen; length bar = 100 μm. (c,d) Whole lung homogenates were analyzed on day 14 for acid-soluble collagen using the Sircol assay (c), and on day 21 for hydroxyproline content (d). Values represent mean± S.E.M.; n = 4–6; *P < 0.01 compared to saline, nontargeting (control) siRNA.

Comment in

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