NOX4/NADPH oxidase expression is increased in pulmonary fibroblasts from patients with idiopathic pulmonary fibrosis and mediates TGFbeta1-induced fibroblast differentiation into myofibroblasts

Abstract

Background: Persistence of myofibroblasts is believed to contribute to the development of fibrosis in idiopathic pulmonary fibrosis (IPF). Transforming growth factor beta1 (TGFbeta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (alpha-SMA) and produce extracellular matrix proteins, such as procollagen I (alpha1). Reactive oxygen species produced by NADPH oxidases (NOXs) have been shown to regulate cell differentiation. It was hypothesised that NOX could be expressed in parenchymal pulmonary fibroblasts and could mediate TGFbeta1-stimulated conversion of fibroblasts into myofibroblasts.

Methods: Fibroblasts were cultured from the lung of nine controls and eight patients with IPF. NOX4, alpha-SMA and procollagen I (alpha1) mRNA and protein expression, reactive oxygen species production and Smad2/3 phosphorylation were quantified, in the absence and in the presence of incubation with TGFbeta1. Migration of platelet-derived growth factor (PDGF)-induced fibroblasts was also assessed.

Results: It was found that (1) NOX4 mRNA and protein expression was upregulated in pulmonary fibroblasts from patients with IPF and correlated with mRNA expression of alpha-SMA and procollagen I (alpha1) mRNA; (2) TGFbeta1 upregulated NOX4, alpha-SMA and procollagen I (alpha1) expression in control and IPF fibroblasts; (3) the change in alpha-SMA and procollagen I (alpha1) expression in response to TGFbeta1 was inhibited by antioxidants and by a NOX4 small interfering RNA (siRNA); (4) NOX4 modulated alpha-SMA and procollagen I (alpha1) expression by controlling activation of Smad2/3; and (5) NOX4 modulated PDGF-induced fibroblast migration.

Conclusion: NOX4 is critical for modulation of the pulmonary myofibroblast phenotype in IPF, probably by modulating the response to TGFbeta1 and PDGF.

Figure 1

A, B, C and D: NOX1, 2, 4 and 5 mRNA expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) compared to controls expressed as the ratio to ubiquitin mRNA levels. Data are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values. E: Typical western-blot and quantification of NOX4 protein expression (67 kD) in lung fibroblasts from patients with IPF and controls. NOX4 is expressed as the ratio to β-actin expression. Data are presented as box-and- whiskers plot with median, interquartile range and minimum and maximum values. F: reactive oxygen species production by IPF and control fibroblasts assessed by measurement of H₂-DCFH-DA oxidation. The values, expressed as arbitrary fluorescence units, are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values.

Figure 2

Detection of immunoreactive NOX-4 in the lung. Immunohistochemistry shows that bronchial and alveolar epithelial cells express NOX-4 in the normal lung. In IPF lung samples, hyperplastic alveolar cells and fibroblasts (arrows) are strongly labelled. The control antibody gave always a negative signal.

Figure 3

A and B: Effect of TGF-β1 in the presence or in the absence of N-acetylcysteine (NAC, 1 mM) or diphenylene iodonium (DPI, 10 μm) on NOX4, α smooth muscle actin (α-SMA) and procollagen I (α1) mRNA expression in lung fibroblasts from controls and patients with idiopathic pulmonary fibrosis (IPF). Data are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values. C: Typical western blot and quantification of α-SMA protein expression in lung fibroblasts after TGF-β1 incubation in the presence or in the absence of NAC (1 mM) or DPI (10 μm). α-SMA is expressed as the ratio to β-actin expression. Data are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values. D: TGF-β1 stimulates ROS production in human lung fibroblasts. Control and IPF fibroblasts were stimulated with TGF-β1 for 1 to 24 h and ROS production was assessed by measurement of H₂-DCFH-DA oxidation. The 4 figures show production by the 2 groups of cells after 1, 3 12 and 24 h post-TGF-β1 respectively. Values are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values.

Figure 3

A and B: Effect of TGF-β1 in the presence or in the absence of N-acetylcysteine (NAC, 1 mM) or diphenylene iodonium (DPI, 10 μm) on NOX4, α smooth muscle actin (α-SMA) and procollagen I (α1) mRNA expression in lung fibroblasts from controls and patients with idiopathic pulmonary fibrosis (IPF). Data are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values. C: Typical western blot and quantification of α-SMA protein expression in lung fibroblasts after TGF-β1 incubation in the presence or in the absence of NAC (1 mM) or DPI (10 μm). α-SMA is expressed as the ratio to β-actin expression. Data are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values. D: TGF-β1 stimulates ROS production in human lung fibroblasts. Control and IPF fibroblasts were stimulated with TGF-β1 for 1 to 24 h and ROS production was assessed by measurement of H₂-DCFH-DA oxidation. The 4 figures show production by the 2 groups of cells after 1, 3 12 and 24 h post-TGF-β1 respectively. Values are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values.

Figure 4

A and B: Effect of cell transfection with a siRNA anti-NOX4 on NOX4, α-SMA and procollagen I (α1) mRNA expression induced by TGF-β1 in lung fibroblasts from controls and patients with idiopathic pulmonary fibrosis (IPF). siScr: scrambled siRNA. NOX4, α-SMA and procollagen I (α1) mRNA levels are expressed as the ratio to ubiquitin mRNA levels. Values are presented as box-and-whiskers plot with median, interquartile range and minimum and maximum values.

Figure 5

Typical western bolt showing the expression of phosphorylated (p-Smad 2/3) and non-phosphorylated (Smad 2/3) in human lung fibroblasts from controls and patients with idiopathic pulmonary fibrosis (IPF) after incubation with TGF-β1 in the presence or in the absence of a scrambled siRNA (siScr), a siRNA anti-NOX4 (siNOX4), diphenylene iodonium (DPI, 10 μm) or N-acetylcysteine (NAC, 1 mM). Box-and-whiskers plot with median, interquartile range and minimum and maximum values show quantification of p-Smad 2/3 expressed as the ratio of Smad 2/3 expression.