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The transcription factor NRF2 protects against pulmonary fibrosis

HYE-YOUN CHO^*,,1, SEKHAR P. M. REDDY^*, MASAYUKI YAMAMOTO and STEVEN R. KLEEBERGER^*,

^* Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA;
Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA; and
Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tennoudai, Tsukuba, Japan

¹Correspondence: Laboratory of Respiratory Biology, NIEHS, NIH, Research Triangle Park, North Carolina 27709, USA. E-mail: cho2{at}niehs.nih.gov

SPECIFIC AIMS

Molecular mechanisms of fibrosis are poorly understood, although reactive oxygen species (ROS) are thought to have an important role. The primary objective of this study was to determine whether NF-E2-related factor 2 (NRF2), a key transcriptional regulator for antioxidant response element (ARE) -mediated induction of cellular antioxidants and detoxifying proteins, protects against pathogenesis of pulmonary fibrosis. To test this hypothesis, we exposed mice with targeted deletion of Nrf2 (ICR/Sv129-Nrf2^–/–) and wild-type (ICR/Sv129-Nrf2^+/+) mice to bleomycin and compared pulmonary injury and fibrotic responses.

PRINCIPAL FINDINGS

1. Effects of targeted disruption of Nrf2 on lung injury and fibrosis phenotypes
Mice (male, 6–10 wk) were anesthetized with 1.39 mg ketamine and 0.22 mg xylozine in saline (0.1 mL, i.p.), and a single dose (1.5 or 3.2 U/kg) of bleomycin in saline (1.25 U/mL) was delivered by intratracheal instillation. An equivalent volume of saline was instilled in control mice of each genotype. A significantly greater increase (40%) in lung/body weight ratio (a parameter of lung edema and matrix deposition) was found in Nrf2^–/– mice compared with Nrf2^+/+ mice 6 days after bleomycin (3.2 U/kg). Bleomycin (3.2 U/kg) significantly increased (33%) lung hydroxyproline content (a marker of collagen deposition) over Nrf2^–/– vehicle controls; no changes were found in Nrf2^+/+ mice. The lower dose of bleomycin (1.5 U/kg) was used to investigate prefibrotic molecular and cellular events as well as time-dependent changes in pathology. Fourteen days after bleomycin (1.5 U/kg) instillation, a significantly greater loss of body weight was found in Nrf2^–/– mice, relative to Nrf2^+/+ mice. Mean numbers of total cells, neutrophils, lymphocytes, and epithelial cells recovered in bronchoalveolar lavage fluid were significantly higher (2 to 3-fold) in Nrf2^–/– mice than in Nrf2^+/+ mice 14 days after low dose (Fig. 1 ) and 6 days after high dose bleomycin (data not shown). Compared with Nrf2^+/+ mice, lung collagen accumulation determined by Sirius red dye-collagen binding assay was significantly greater in Nrf2^–/– mice 3, 7, and 14 days after bleomycin treatment. Statistically significant deposition of collagen in Nrf2^+/+ mice was found only at 14 days after bleomycin.

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of targeted disruption of Nrf2 on bleomycin (1.5 U/kg)-induced increases in bronchoalveolar lavage phenotypes 14 days after instillation. Data are presented as means ± SEM (n=4–5/group). *Significantly different from genotype-matched vehicle control mice (P<0.05). +Significantly different from bleomycin-treated Nrf2+/+ mice (P<0.05).

2. Effects of targeted disruption of Nrf2 on pulmonary pathology
Histological staining (Sirius red/Fast green, H&E) of lung tissue sections indicated significant differences in progression of pulmonary injury, inflammation, and fibrosis between Nrf2^–/– and Nrf2^+/+ mice (Fig. 2 ). Predominant histopathologic features of Nrf2^–/– mice instilled with bleomycin (1.5 U/kg) included alveolar inflammation (principally neutrophils and lymphocytes), diffused fibrotic patch formation with collagen accumulation, hyperplastic and hypertrophic changes of epithelium lining alveoli, small bronchi, and terminal bronchioles. Fibrogenesis was observed primarily in alveolar interstitium adjacent to terminal bronchioles, and alveolar bronchiolization was distinct in areas undergoing severe fibrotic remodeling. Compared with Nrf2^–/– mice, relatively mild inflammation and only focal fibrotic lesions were found in Nrf2^+/+ mice at 7 days. Persistent inflammation and abnormal lung architecture characterized by extensive fibroblast proliferation, alveolar bronchiolization, and collagen-stained matrix deposition was evident in Nrf2^–/– mice 14 days after instillation. Fibrosis scoring by Ashcroft method ranging from 0 (no fibrosis) to 8 (total fibrosis) demonstrated that significant lung fibrosis was evident at 7 and 14 days postinstillation in both genotypes of mice. However, average fibrotic scores were significantly higher in Nrf2^–/– mice compared with Nrf2^+/+ mice at 7 days (60%; 4.98±0.32 vs. 3.16±0.33) and 14 days (27%; 6.77±0.46 vs. 5.31±0.45) after bleomycin.

View larger version (79K): [in this window] [in a new window]   Figure 2. Differential progression of pulmonary injury and fibrosis in Nrf2+/+ (A–D) and Nrf2–/– (E–H) mice after bleomycin (1.5 U/kg). Paraffin-embedded left lung tissue sections were processed for histological analyses and Sirius red/Fast green staining was performed to visualize collagen deposition (red dye staining) in control lungs (A, E) or fibrotic lung interstitium 7 (B, F) or 14 days (C, G) after bleomycin. Higher magnification of H&E-stained lung tissue sections illustrates alveolar bronchiolization in fibrotic lesions of Nrf2+/+ (D) and Nrf2–/– (H) mice 14 days after bleomycin. Arrows, bronchiolization; TB, terminal bronchiole; BR, bronchiole; BV, blood vessel. Bar: 100 µm.

3. Bleomycin-Induced mRNA/Protein expression and transactivation of lung NRF2
Bleomycin caused significant time-dependent induction of NRF2 mRNA expression in lungs of Nrf2^+/+ mice (50% over saline controls at 14 days). Immunoblotting analyses detected marked bleomycin-induced increases of cytoplasmic and nuclear NRF2 accumulation in lungs of Nrf2^+/+ mice (14 days after instillation). Time-dependent increase of bleomycin-induced nuclear NRF2-DNA (NF-E2) binding activity was detected by gel mobility shift/supershift analysis in lungs of Nrf2^+/+ mice.

4. Effects of targeted disruption of Nrf2 on bleomycin-induced antioxidant enzyme expression
Bleomycin caused up-regulation of mRNA for NADP(H):quinone oxidoreductase (NQO1), glutathione-S-transferase (GST)-Ya ()/-Yp1 (pi)/-Yb1 (mu), catalytic (GLCLc) and regulatory (GLCLr) subunits of gamma glutamylcysteine ligase, thioredoxin reductase (TXNRD) 1, UDP glycosyl transferase 1a6, carboxylesterase (Ex), heme oxygenase (HO)-1, glutathione peroxidase (GPx) 2, and superoxide dismutase (SOD)-3 in Nrf2^+/+ mice over basal levels. Bleomycin-induced expression of all these genes was significantly higher in Nrf2^+/+ mice than in Nrf2^–/– mice. Among these, mRNA levels of NQO1, TXNRD1, GLCLc, Ex, and GPx2 were constitutively attenuated in Nrf2^–/– mice compared with Nrf2^+/+ mice, and their steady-state expression levels were not significantly altered by bleomycin instillation in Nrf2^–/– mice. Consistent with gene expression results, bleomycin-induced protein levels of pulmonary GST-, GST-mu, GPx, and HO-1 were significantly greater in Nrf2^+/+ mice than in Nrf2^–/– mice, with peaks at 7 days. No statistically significant changes were detected in Nrf2^–/– mice after bleomycin.

5. Effects of targeted disruption of Nrf2 on bleomycin-induced lung injury/fibrosis marker expression
Greater accumulation of transcripts for several matrix markers of fibroproliferation (type 1 collagen 2; Col1a2, fibronectin 1; FN1, tenascin-C) and pulmonary injury and remodeling markers (transforming growth factor-ßs; TGF-ßs, matrix metalloproteinases; MMPs, tissue inhibitor of metalloproteinase 1; TIMP1) was found in Nrf2^–/– mice compared with Nrf2^+/+ mice after bleomycin. Transcripts of many of these genes were constitutively higher in Nrf2^–/– mice than in Nrf2^+/+ mice, while tenascin-C and MMP7 were not detected in control lung tissues. Consistent with gene expression results, protein levels of lung MMP2, TGF-ß, connective tissue growth factor (CTGF), and tenascin-C were significantly higher in Nrf2^–/– mice than in Nrf2^+/+ mice after instillation (7 or 14 days postinstillation).

CONCLUSIONS AND SIGNIFICANCE

Results of the present study supported the hypothesis that NRF2 is protective against fibrotic lung injury. Relative to Nrf2^+/+ mice, targeted deletion of Nrf2 significantly enhanced susceptibility to murine pulmonary inflammation and fibrosis induced by the anti-neoplastic drug bleomycin. Suppressed induction of NRF2-dependent antioxidant/defense enzymes in lungs of Nrf2^–/– mice suggests that these enzymes may contribute to NRF2-mediated protection against bleomycin-induced lung fibrosis. This is the first study to suggest an upstream regulatory mechanism of cellular antioxidant enzyme defense in an experimental fibrosis model.

Fibrosis is an increasingly prevalent disorder, and is an end-state process in a number of chronic diseases of kidney, liver, and lung. In particular, idiopathic pulmonary fibrosis is a progressive and usually fatal disease of unknown etiology with no known effective therapy. A role for oxidants in the pathogenesis of pulmonary fibrosis has been suggested in previous studies. Protective roles of antioxidative mechanisms in pulmonary fibrosis were demonstrated by examining the role of enzymatic (e.g., SODs) or nonenzymatic (e.g., N-acetylcysteine) antioxidants. The thiol redox system (glutathione, thioredoxin) was also determined to be protective against lung fibrosis. The present study supports this concept, and provides a novel molecular mechanism through which antioxidative protection against fibrogenesis may occur. It is, however, important to note that antioxidative mechanism mediated by ARE-NRF2 response was not sufficient to completely ameliorate inflammation and fibrotic responses to bleomycin. Other antifibrotic mechanisms (e.g., interleukins-9 and 12) may interact for protection against fibrosis.

The present study demonstrated that bleomycin caused increased mRNA expression, lung protein levels, and nuclear DNA binding of NRF2 in advance of the onset of pulmonary inflammation and fibrogenesis. As depicted in Fig. 3 , our findings suggest that activation of NRF2 and ARE-mediated induction of antioxidant defense enzymes during pathogenesis of ROS-mediated fibrogenesis has a key role in combating ROS and suppression of fibrotic tissue injury. Results from the current study may have important implications for development of combined therapies for bleomycin-toxicity and idiopathic pulmonary fibrosis to complement anti-inflammation therapy, which is only effective in subsets of fibrosis patients.

View larger version (47K): [in this window] [in a new window]   Figure 3. A hypothetical mechanism depicts a protective role of NRF2 in the pathogenesis of pulmonary fibrosis. Imbalance of cellular oxidant (ROS) and of antioxidant capacity induced by injury and inflammation causes oxidative stress in pulmonary cells. It may cause prefibrotic events (e.g., activation of growth factors) leading to matrix deposition and fibroblast proliferation. Oxidative stress may also trigger modification (e.g., phosphorylation) of Keap1-NRF2 sensor to release NRF2 from the complex. NRF2 binding to ARE in association with other transcription factor (e.g., small Maf, c-Jun) can induce transcriptional activation of antioxidant/detoxifying proteins to combat against ROS and further oxidative injury.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1127fje; doi: 10.1096/fj.03-1127fje

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