Nrf2 is a master transcription activator of cytoprotective genes through antioxidant response element (ARE) binding. Hyperoxia exposure to laboratory rodents induces pulmonary injury which resembles acute lung injury (ALI) phenotypes. Genome-wide linkage analysis in mice revealed Nrf2 as a pulmonary hyperoxia susceptibility gene, and its protective roles have been determined. The current study was designed to discover sequence variation in mouse Nrf2 and to determine its association with hyperoxia susceptibility in inbred strains. Single nucleotide polymorphisms (SNPs) in Nrf2 genome including 5 kb upstream promoter were compiled for 66 inbred strains from publicly available data base. Biallelic matrix mapping of the SNPs categorized the mice into 3 distinct haplotypes: 27 strains, haplotype 1;23 strains, haplotype 2, and 3 strains, haplotype 3. Nrf2 from selected 16 strains of the 3 haplotypes were re-sequenced. A total of 1,110 SNPs included 162 in 5 upstream, 48 in exons, 779 in introns, and 121 in 3 downstream regions. Sixteen strains were exposed to hyperoxia (>95% O2), and lung injury phenotypes and Nrf2-ARE responses were compared. Functional roles of SNP haplotypes were determined in vitro by comparing pulmonary expression and activity of Nrf2 variants. Hyperoxia-induced body weight loss and lung injury was relatively greater in strains of Haplotypes 2 and 3 than Haplotype 1. The -103T/C SNP which adds a Sp1 binding site in Haplotype 2 suppressed the hyperoxia-induced promoter activation. Nrf2 from Haplotype 3 mice bearing non-synonymous SNPs located in (1862A>T, His543Gln) and adjacent to (1417T>C, Thr395Ile) Neh1 domain showed lowered nuclear transactivation after hyperoxia than Haplotypes 1 Nrf2. Results indicate that murine Nrf2 is polymorphic, and correlation of haplotypes and phenotypes further supports Nrf2 as a susceptibility gene in hyperoxic lung injury. Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like;NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells'SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis. Oxidants have been proposed to contribute to the development chronic pulmonary disorders including bronchopulmonary dysplasia (BPD). Little is known about the role of genetic background in susceptibility to BPD phenotypes in neonates. Support for a genetic contribution to BPD susceptibility developed as variation in frequency and severity of BPD in preterm infants having similar environmental risk factors was reported. A twin study reported that genetics accounted for 79-82% variation in human BPD. To develop a genetic model of differential susceptibility to BPD, we phenotyped 34 neonatal inbred strains of mice at post-natal ages P1-P4 in the late saccular stage of lung development for BPD phenotypes in response to hyperoxia. We found significant inter-strain variation in BAL inflammation and injury phenotypes with heritability indices ranging 33.6-55.7%. Interestingly, the strain distribution patterns for 8hyperoxia response phenotypes are different from those for strain-matched adults, i.e. we did not recapitulate in neonates what was known previously for adults. For example, C3 mice are among the most susceptible neonates, but are the most resistant adults. This suggests that susceptibility mechanisms differ between adults and neonates and/or interaction with lung growth in the neonates is an important co-factor for hyperoxic lung injury. In collaboration with Dr. Tim Wiltshire (UNC) haplotype association mapping identified multiple associations with significant logP scores for BAL inflammation and injury phenotypes. Significant QTLs included chromosomes 1, 2, 7, 4, 5, and 6, and potential candidate susceptibility genes in these QTLs have been identified. Interestingly, chromosomal regions identified for neonate susceptibility did not overlap with those for adults, consistent with discordance of phenotypes between neonates and adults. We also identified a number of interesting candidate genes in the QTLs, including Chrm2 (cholinergic receptor, muscarinic 2, cardiac). Further, hyperoxia-induced lung injury was significantly reduced in neonatal mice with targeted deletion of Chrm2, relative to wild-type controls. This approach is an important first step to understand the genetic basis of susceptibility to lung injury, and has been validated for a number of complex traits. Another study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mice. Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2(-/-) neonates tthan in Nrf2(+/+) neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2(-/-) neonates than in Nrf2(+/+) neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell-cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors. This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs. Together this study indicated that Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD.

Project Start
Project End
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Support Year
12
Fiscal Year
2013
Total Cost
$606,752
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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
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
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
Reddy, Narsa M; Suryanarayana, Vegiraju; Kalvakolanu, Dhananjaya V et al. (2009) Innate immunity against bacterial infection following hyperoxia exposure is impaired in NRF2-deficient mice. J Immunol 183:4601-8
Reddy, Narsa M; Suryanaraya, Vegiraju; Yates, Melinda S et al. (2009) The triterpenoid CDDO-imidazolide confers potent protection against hyperoxic acute lung injury in mice. Am J Respir Crit Care Med 180:867-74
Reddy, Narsa M; Kleeberger, Steven R; Kensler, Thomas W et al. (2009) Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice. J Immunol 182:7264-71
Reddy, Anita J; Christie, Jason D; Aplenc, Richard et al. (2009) Association of human NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphism with development of acute lung injury. J Cell Mol Med 13:1784-91
Cho, Hye-Youn; Kleeberger, Steven R (2009) Nrf2 protects against airway disorders. Toxicol Appl Pharmacol :

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