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. In these same strains of mice, we are also mapping the QTLs that associate with differential susceptibility to oxidant-induced nuclear and mitochondrial lesions, as well as mitochondrial copy number. Moreover, we have initiated an investigation to ultra-deep sequence the mitochondrial genome in strains of mice with normoxia and hyperoxia to identify sequence variations and heterplasmies that may be induced by these exposures. The role of Nrf2 on heart rate (HR) and heart rate variability (HRV) responses is not known. We hypothesized that genetic disruption of Nrf2 would exacerbate murine HR and HRV responses to severe hyperoxia or moderate PM exposures. Nrf2(-/-) and Nrf2(+/+) mice were instrumented for continuous ECG recording to calculate HR and HRV (low frequency (LF), high frequency (HF), and total power (TP)). Mice were then either exposed to hyperoxia for up to 72 hrs or aspirated with ultrafine PM (UF-PM). Compared to respective controls, UF-PM induced significantly greater effects on HR (P < 0.001) and HF HRV (P < 0.001) in Nrf2(-/-) mice compared to Nrf2(+/+) mice. Nrf2(-/-) mice tolerated hyperoxia significantly less than Nrf2(+/+) mice (22 hrs; P < 0.001). Reductions in HR, LF, HF, and TP HRV were also significantly greater in Nrf2(-/-) compared to Nrf2(+/+) mice (P < 0.01). Results demonstrate that Nrf2 deletion increases susceptibility to change in HR and HRV responses to environmental stressors and suggest potential therapeutic strategies to prevent cardiovascular alterations.
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