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. 2013 Jan 11;288(2):770-7.
doi: 10.1074/jbc.M112.431973. Epub 2012 Nov 30.

Mitochondrial reactive oxygen species regulate transforming growth factor-β signaling

Manu Jain  1 , Stephanie Rivera, Elena A Monclus, Lauren Synenki, Aaron Zirk, James Eisenbart, Carol Feghali-Bostwick, Gokhan M Mutlu, G R Scott Budinger, Navdeep S Chandel
Affiliations

Mitochondrial reactive oxygen species regulate transforming growth factor-β signaling

Manu Jain et al. J Biol Chem. .

Abstract

TGF-β signaling is required for normal tissue repair; however, excessive TGF-β signaling can lead to robust profibrotic gene expression in fibroblasts, resulting in tissue fibrosis. TGF-β binds to cell-surface receptors, resulting in the phosphorylation of the Smad family of transcription factors to initiate gene expression. TGF-β also initiates Smad-independent pathways, which augment gene expression. Here, we report that mitochondrial reactive oxygen species (ROS) generated at complex III are required for TGF-β-induced gene expression in primary normal human lung fibroblasts. TGF-β-induced ROS could be detected in both the mitochondrial matrix and cytosol. Mitochondrially targeted antioxidants markedly attenuated TGF-β-induced gene expression without affecting Smad phosphorylation or nuclear translocation. Genetically disrupting mitochondrial complex III-generated ROS production attenuated TGF-β-induced profibrotic gene expression. Furthermore, inhibiting mitochondrial ROS generation attenuated NOX4 (NADPH oxidase 4) expression, which is required for TGF-β induced myofibroblast differentiation. Lung fibroblasts from patients with pulmonary fibrosis generated more mitochondrial ROS than normal human lung fibroblasts, and mitochondrially targeted antioxidants attenuated profibrotic gene expression in both normal and fibrotic lung fibroblasts. Collectively, our results indicate that mitochondrial ROS are essential for normal TGF-β-mediated gene expression and that targeting mitochondrial ROS might be beneficial in diseases associated with excessive fibrosis.

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Figures

FIGURE 1.
FIGURE 1.
TGF-β induces mitochondrial ROS which is inhibited by a mitochondrially targeted antioxidant. A, primary cultures of NHLFs were treated with TGF-β (5 ng/ml) in the presence or absence of SB431542. B, primary cultures of NHLFs were treated with TGF-β (5 ng/ml) alone and in the presence of TPP (1 μm) or MitoQ (1 μm), and intracellular H2O2 levels were assessed using Amplex Red. C, primary NHLFs were infected with 100 pfu of adenovirus encoding mito-roGFP. Twenty-four hours after infection, the cells were treated with TGF-β1 (5 ng/ml) or antimycin A (1 μg/ml) for 1 h, and oxidation of the probe was measured by flow cytometry. D, primary NHLFs were infected with 100 pfu of adenovirus encoding cyto-roGFP. Twenty-four hours after infection, the cells were treated with TGF-β1 (5 ng/ml) or antimycin A (1 μg/ml) for 1 h, and oxidation of the probe was measured by flow cytometry. E, primary NHLFs were infected with 100 pfu of adenovirus encoding mito-roGFP. Twenty-four hours after infection, the cells were treated with TGF-β (5 ng/ml) in the presence of TPP (1 μm) or MVE (1 μm, 1-h pretreatment), and oxidation of the probe was measured by flow cytometry. F, primary NHLFs were infected with 100 pfu of adenovirus encoding cyto-roGFP. Twenty-four hours after infection, the cells were treated with TGF-β1 (5 ng/ml) in the presence of TPP (1 μm) and MVE (1 μm, 1-h pretreatment), and oxidation of the probe was measured by flow cytometry. Error bars represent mean ± S.E. (n = 3 for A–D and n = 6 for E and F). *, p < 0.05 for comparison between TGF-β and the control; †, p < 0.05 for comparison between MVE and TPP.
FIGURE 2.
FIGURE 2.
Mitochondrially targeted antioxidants inhibit TGF-β-mediated gene transcription downstream of the nuclear translocation of phosphorylated Smad3. A, primary NHLFs were treated with TGF-β (5 ng/ml) in the presence or absence of MVE or its control cation (TPP) (both 1 μm), and cytosolic and nuclear fractions were isolated and immunoblotted using antibodies to phosphorylated (p-Smad3) and total Smad3. Antibodies to actin and RNA polymerase II (RNA pol II) were used as loading controls and to ensure exclusion of cytosolic proteins. B, primary NHLFs were treated with TGF-β (5 ng/ml) with or without MVE or TPP (both 1 μm), and 24 h later, cell death was measured by propidium iodine staining. C and D, NHLFs were transfected with a plasmid containing SBE-luciferase and treated 24 h later with TGF-β (5 ng/ml) with or without MitoQ (C), MVE (D), or TPP (all 1 μm), and SBE-luciferase activity was measured 24 h later. E and F, primary NHLFs were grown to 70% confluence and incubated with TGF-β (5 ng/ml) with or without MitoQ (E), MVE (F), or TPP, and 24 h later, the levels of mRNAs encoding α-SMA, CTGF, and NOX4 were measured using quantitative RT-PCR in cell lysates. Error bars represent mean ± S.E. (n = 3 for all measures). *, p < 0.05 for comparison between TGF-β1 and the control; †, p < 0.05 for comparison between MitoQ or MVE and TPP.
FIGURE 3.
FIGURE 3.
Mitochondrially generated ROS are necessary for TGF-β-mediated transcription. A and B, primary NHLFs were stably transfected with a shRNA against RISP, QPC, or control lentiviruses (Drosophila hypoxia-inducible factor and pLKO), and the levels of RISP and QPC were measured by immunoblotting. C and D, these cells were treated with TGF-β, and intracellular H2O2 levels were measured using Amplex Red. E and F, control and RISP and QPC knockdown fibroblasts were treated with TGF-β (5 ng/ml), and 24 h later, mRNAs encoding the TGF-β target genes α-SMA, CTGF, and NOX4 were measured by quantitative RT-PCR. Error bars represent mean ± S.E. (n = 3 for all measures). *, p < 0.05 for comparison between TGF-β and the control; †, p < 0.05 for comparison between RISP knockdown and the control.
FIGURE 4.
FIGURE 4.
Mitochondrial ROS are sufficient to augment TGF-β-mediated transcription in primary NHLFs. A, primary NHLFs were stably transfected with a shRNA encoding QPC or a control lentivirus and treated with TGF-β (5 ng/ml) in the presence or absence of MVE, and 24 h later, the levels of mRNAs encoding the TGF-β transcriptional targets α-SMA, CTGF, and NOX4 were measured by quantitative RT-PCR. kd, knockdown. B and C, primary NHLFs were treated with antimycin A (1 μm), which inhibits electron transport through cytochrome b (analogous to the loss of QPC), with or without TGF-β (5 ng/ml) (B) and with or without MVE (C), and 24 h later, the levels of mRNAs encoding the same TGF-β transcriptional targets were measured. Error bars represent mean ± S.E. (n = 3 for all measures). *, p < 0.05 for comparison between TGF-β and the control; †, p < 0.05 for comparison between antimycin A and the control; ‡, p < 0.05 for comparison between MVE and the control.
FIGURE 5.
FIGURE 5.
Mitochondrial ROS are required for TGF-β-induced gene expression in lung fibroblasts obtained from patients with lung fibrosis and scleroderma. A, primary cultures of lung fibroblasts from each of four patients with lung fibrosis were treated with TGF-β (5 ng/ml), and 30 min later, intracellular H2O2 levels were measured using Amplex Red. Values are presented as the -fold change compared with cultures of NHLFs. B and C, primary lung fibroblasts cultured from two patients with scleroderma-associated pulmonary fibrosis were treated with TGF-β (5 ng/ml) in the presence or absence of Mito-CP or its control cation (TPP), and 24 h later, the levels of mRNAs encoding α-SMA and NOX4 were measured by quantitative RT-PCR. D and E, the experiments were repeated with lung fibroblasts from two patients with idiopathic pulmonary fibrosis. Error bars represent mean ± S.E. (n = 3 for all measures). *, p < 0.05 for comparison between fibroblasts from patients with lung fibrosis and NHLFs; †, p < 0.05 for comparison between TGF-β and the control; ‡, p < 0.05 for comparison between Mito-CP and TPP. SSc, systemic sclerosis (scleroderma); IPF, idiopathic pulmonary fibrosis.
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