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). 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 indicated increased risk for BPD in twin pairs independent of other factors such as gestational age and gender (74). Another twin study reported that genetics accounted for 79-82% variation in BPD and Bhandari et al. calculated a heritability of 53% after controlling for covariates. Preterm infants are also at greater risk for developing BPD in cases with maternal diabetes, birth asphyxia, and familial asthma. Polymorphisms in many genes involved in lung development, inflammation, and vascularization have been considered in BPD including SP-A and SP-B, VEGF, IL-1, IL-10, TGF-, IGF, MCP-1, ACE, and TNF-a. 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% (Fig 13). Interestingly, the strain distribution patterns for hyperoxia 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. Vancza et al also found age-dependent effects following O3 exposure and, as with hyperoxia, C3 neonates were very susceptible to O3 as neonates but resistant as adults. In collaboration with Dr. Tim Wiltshire (UNC) following procedures of Pletcher et al, HAM identified multiple associations with significant logP scores for BAL inflammation and injury phenotypes. Significance thresholds for each analysis were defined by determining the false positive rate of each p value. 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 (44), consistent with discordance of phenotypes between neonates and adults. 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 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.
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