NRF2 protein lacks the capability to bind DNA, therefore requires a partner to bind regulatory elements. These binding partners include small Maf basic leucine zipper proteins, MAFF, MAFK, and MAFG. Maf proteins have redundant functionality, and expression varies by both cell type and stimulus. We have begun to focus on the antioxidant role of MAFG in bronchial epithelial cells. Using the normal-derived airway cell line, BEAS-2B, we demonstrate that MAFG gene expression levels are approximately 9-fold or greater than MAFK or MAFF levels, based on quantitative PCR. Silencing of MAFG gene expression significantly enhanced cellular cytotoxicity to juglone, a potent superoxide generator, compared to controls. Additionally we performed an Illumina whole-genome microarray on MAFG- or NRF2-silenced BEAS-2B. To induce NRF2-mediated gene expression, we also treated cells with sulforaphane, which robustly activates NRF2 (as evidenced by increased NRF2 binding to consensus sequence). Of the 504 genes whose expression was significantly modulated by 4h 10M sulforaphane treatment, 44 were altered with both MAFG and NRF2 silencing, suggesting that MAFG regulates these genes via the NRF2 pathway. To support these data, we examined transcript levels in normal bronchial epithelial cells of lung cancer patients (Spira et al., Nat. Med. 2007.). Of note, MAFG appears to be underexpressed in the airways of smokers who later develop lung cancer. Our findings suggest that reduction in MAFG compromises the capability of the epithelial airway to neutralize and respond oxidative stress. Oxidants have been proposed to contribute to the development chronic pulmonary disorders including bronchopulmonary dysplasia (BPD). The objective of this new investigation is to identify genetic factor(s) that render differential susceptibility of neonatal inbred strains of mice to BPD phenotypes caused by hyperoxia. Neonates pooled from multiple litters of each inbred strain (P1) were randomly assigned and exposed to hyperoxia (100% O2) or air for 3d with foster dams. Lung injury phenotypes were determined by bronchoalveolar lavage (BAL) and histopathologic analysis. Inter-strain variance was significantly greater than the intra-strain variance in pulmonary inflammatory and injury responses found among the 25 strains studied. The strain distributions for increases in total BAL cell numbers (epithelial and inflammatory cells) and lung permeability were continuous, and C57BL/6J mice were among the most susceptible and BALB/CBYJ and C3H/HeJ mice were among the most resistant. Histopathological analysis of H&E-stained lung sections also demonstrated variations in the magnitude of hyperoxia-induced edema in air spaces and perivascular/peribronchial regions, alveolar and bronchiolar proliferation, and inflammatory cell infux among the strains. Broad sense heritability estimates of the pulmonary response to exposure was high, but varied across phentoypes. The inter-strain variation suggests a strong genetic component(s) in BPD pathogenesis in mice. Positional cloning analysis will identify quantitative trait loci (QTLs) and candidate gene(s) for neonatal BPD phenotypes in mice. Another investigation was designed to test the hypothesis that lack of Nrf2-mediated antioxidant pathway enhances the BPD phenotypes caused by hyperoxia. Nrf2-deficient (Nrf2-/-) and wild-type (Nrf2+/+) neonatal mice (P1) were exposed to hyperoxia (O2) or air with foster dams. Lung injury phenotypes were determined in both genotypes by bronchoalveolar lavage (BAL) analyses and histopathology. Pulmonary Nrf2 and antioxidant levels as well as lung oxidation status were also compared. O2 (100%) induced significant lung neutrophilia and cell death in Nrf2+/+ pups at 3 d. The magnitude of O2 sensitivity and lung injury parameters including mortality, suppressed body weight gain, lung edema and inflammation, and BAL LDH level were significantly higher in Nrf2-/- pups than in Nrf2+/+ pups. Nrf2-/- mice displayed greater lung protein oxidation levels than Nrf2+/+ pups basally and after O2. Hyperoxia-induced activation of nuclear Nrf2 and induction of ARE-responsive lung antioxidant enzymes were suppressed in Nrf2-/- pups compared to Nrf2+/+ mice. Neither mouse genotype significantly responded to low dose (70%) hyperoxia by 6 d though body weight gain was significantly attenuated in Nrf2-/- pups (70%) compared to Nrf2+/+ pups (90%) relative to the corresponding air controls. Results support an essential protective role for Nrf2 in BPD pathogenesis in developing lung. We have also begun global gene expression analyses in developing lungs from Nrf2+/+ and Nrf2-/- neonates. The current study was designed to identify gene expression pathways that define the role of Nrf2 in lung development, and to identify Nrf2-mediated events that may have implications on exposure-related events later in life. Gene expression profiles were characterized in lungs from Nrf2-/- and Nrf2+/+ mouse neonates at P1-P4, or after exposure to hyperoxia or air (1-3 d) from P1 using Affymetrix gene arrays. In Nrf2+/+ pups, genes significantly increased at P2-P4 over P1 included genes involved in cellular assembly, organization, and proliferation. Genes encoding channel/junction, carrier, and antioxidant proteins were relatively suppressed in P2-P4 vs. P1 pups. In P1-P4 Nrf2-/- pups, antioxidants, stress response, cell cycle, angiogenesis, and immune genes were suppressed, while Itga4, H2-D1, Jag1, and Trim68 expression was higher than in Nrf2+/+ pups. After hyperoxia, genes varied between Nrf2+/+ and Nrf2-/- mice encode proteins for antioxidants, cell growth/proliferation, angiogenesis/organogenesis, and DNA/protein process, immunity, oncogene, transport, and oxidation. Ingenuity pathway analysis suggested that lack of Nrf2 modifies drug metabolism, cell cycle, and cell-cell signaling/interactions in developing lung, and has a great impact on oxidant scavenging, cancer/immune disease development, lipid metabolism, and cell/organ morphology after hyperoxia. Results provide putative molecular mechanisms of Nrf2-directed lung maturation and oxidative disorders. In another investigation to determine the mechanisms through which hyperoxia exposures affect cardiovascular function, we evaluated the heart rate and QT interval responses to hyperoxia in Bmp 2 and Bmp 4 heterozygous mice. Bone morphogenic proteins (BMPs) are critical in normal development. Bmp2 and Bmp4 have been implicated in embryonic heart development. In mice with hypomorphic Bmp2 or Bmp4 alleles atrioventricular valve and septum defects, respectively, have been reported. We hypothesized that such defects would increase susceptibility to hyperoxia. In this study heart rate (HR) and QT interval (QT) were recorded from mice heterozygous for Bmp2 or Bmp4 and wild-type littermates (WT) during exposure to hyperoxia. Four to six mice per strain (8-12 wks;20-30g) were instrumented with ETA-F20 transmitters. ECG was recorded continuously during exposure to hyperoxia for up to 96 hrs or until moribund. HR and QT (corrected for HR;Bazettes) were assessed before and during the exposure. HRs of the Bmp4+/- and WT, but not of the Bmp2+/-, mice were reduced after 48 hrs of exposure. The slope of the HR responses to hyperoxia suggested Bmp4+/- as the most susceptible strain. Reductions in QT were also observed in WT and Bmp4+/-, but not in the Bmp2+/- mice after 48 hrs of exposure. Despite having the highest QT at baseline, QT in the Bmp4+/- mice remained at this reduced level until moribund. QT of WT mice increased after 48 hrs, above baseline, whereas QT of Bmp2+/- mice decreased after 68 hrs of exposure. These data demonstrate HR response to hyperoxia as a useful predictor of susceptibility;the shorter the time to HR reduction, the more susceptible the strain.

Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2009
Total Cost
$849,363
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Cho, Hye-Youn; Wang, Xuting; Li, Jianying et al. (2016) Potential therapeutic targets in Nrf2-dependent protection against neonatal respiratory distress disease predicted by cDNA microarray analysis and bioinformatics tools. Curr Opin Toxicol 1:125-133
Wang, Xuting; Campbell, Michelle R; Lacher, Sarah E et al. (2016) A Polymorphic Antioxidant Response Element Links NRF2/sMAF Binding to Enhanced MAPT Expression and Reduced Risk of Parkinsonian Disorders. Cell Rep 15:830-842
Cho, Hye-Youn; Jedlicka, Anne E; Gladwell, Wesley et al. (2015) Association of Nrf2 polymorphism haplotypes with acute lung injury phenotypes in inbred strains of mice. Antioxid Redox Signal 22:325-38
Cho, Hye-Youn; Kleeberger, Steven R (2015) Association of Nrf2 with airway pathogenesis: lessons learned from genetic mouse models. Arch Toxicol :
Cho, Hye-Youn; Marzec, Jacqui; Kleeberger, Steven R (2015) Functional polymorphisms in NRF2: implications for human disease. Free Radic Biol Med :
Cho, Hye-Youn; Kleeberger, Steven R (2014) Noblesse oblige: NRF2 functions in the airways. Am J Respir Cell Mol Biol 50:844-7
Nichols, Jennifer L; Gladwell, Wesley; Verhein, Kirsten C et al. (2014) Genome-wide association mapping of acute lung injury in neonatal inbred mice. FASEB J 28:2538-50
Walters, Dianne M; White, Kevin M; Patel, Ushma et al. (2014) Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5). FASEB J 28:1098-112
Messier, Elise M; Day, Brian J; Bahmed, Karim et al. (2013) N-acetylcysteine protects murine alveolar type II cells from cigarette smoke injury in a nuclear erythroid 2-related factor-2-independent manner. Am J Respir Cell Mol Biol 48:559-67
Howden, Reuben; Gougian, Eva; Lawrence, Marcus et al. (2013) The influence of Nrf2 on cardiac responses to environmental stressors. Oxid Med Cell Longev 2013:901239

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