While Nrf2 is a critical modulator for protection against a broad range of oxidative disorders in adults, its role in tissue development or the pathogenesis of neonatal or childhood disease has received little attention. In the current study, transcriptomic analysis indicated that Nrf2 is critical to processes/networks for cell cycle and DNA repair, immune function, morphogenesis and lung development, and antioxidant defense during postnatal normal lung maturation in mice. Importantly, we found a beneficial role for Nrf2 in hyperoxia-induced injury of undeveloped lung. Nrf2
is a susceptibility gene for protection against acute lung injury caused by 100% O2
in adult mice (13
). Our results demonstrate Nrf2-dependent alleviation of hyperoxia-induced injury phenotypes in the saccular phase of lung, including arrest in alveolar development evidenced by lower RAC and reduced appearance of multilobular alveoli/branched septi as well as severe exudative-phase diffuse alveolar damage characterized by edema, leukocyte inflammation, and cell death in Nrf2−/−
mice. Moreover, highly suppressed GSH pools as well as heightened pulmonary oxidation and DNA lesions in Nrf2−/−
neonates indicated the critical roles for ROS and Nrf2-directed defense in the pathogenesis of hyperoxia-induced lung injury. The current study warrants further investigation of Nrf2 in other oxidant-associated lung disease models at early ages. Juvenile Nrf2−/−
mice that were exposed to hyperoxia as neonates had more severely hindered resolution of lung damage relative to juvenile Nrf2+/+
mice that were similarly exposed as neonates (31
). This observation supported an association of the severity of hyperoxia-induced injury in infancy with persisting or long-term pulmonary outcome. It also suggests the potential for exacerbation of oxidative pulmonary disease in adults or adolescents who had BPD in infancy.
The current study initially characterized complex gene expression networks in the saccular stage during postnatal lung maturation. Variation in lung gene expression was greatest at P1 relative to age P4, likely reflecting the influence of direct contact of airway cells to the extra-uterine environment. Importantly, Nrf2
significantly modulated genes involved not only in redox balance but also in tissue and organ development, cancer, cell death, and infectious disease during saccular-to-alveolar transition. The marked overexpression of multiple major histocompatibility complex, class II (MHCII), lymphatic system, and cell–cell interaction genes (e.g.
) in naive lungs from Nrf2−/−
neonates suggested their aberrant basal immunity as evidence by enhanced susceptibility of adult Nrf2−/−
mice to asthma and allergy (28
Our microarray analysis also evaluated Nrf2-dependent antioxidant capacity under normoxic and hyperoxic conditions in the immature lungs. Direct antioxidants, including superoxide dismutases (SODs), are known to be highly activated in the lung shortly after birth (34
), and we found that all the redox genes that varied during P1–P4 were higher at P1 relative to P2–P4. In utero
expression of airway antioxidant enzymes is known to increase toward term gestation to prepare for birth into an O2
-rich (from 3% to 21%) environment (39
). Therefore, preterm infants with low birth weight are not only more sensitive to increased O2
concentrations compared with adults (10
), but they also have diminished/compromised endogenous antioxidant activity relative to full-term infants (39
), which contributes to the critical consequence of hyperoxic insult in BPD pathogenesis. However, overall clinical results from therapies with antioxidants (e.g.
, SODs, vitamins A and E, N-acetylcysteine, and metalloporphyrin) have remained inconclusive in preterm infants (1
). In the current study, we identified novel antioxidants (e.g.
, and Slc7a11
) that were induced during the development of neonatal hyperoxic injury, but not in lungs of adult mice exposed to hyperoxia (15
). These gene products have roles in redox balance through a broad spectrum of pathways, including metabolic process, small molecular biochemistry, and membrane transport. Importantly, Slc7a11
[solute carrier family 7 (cationic amino acid transporter, y+
system), member 11] encodes xCT that is a key component of high-affinity cysteine/glutamate exchange transporter system χc−
, which mediates cellular cystine uptake for GSH synthesis (41
). Identification of putative AREs in these Nrf2 effectors suggests their therapeutic potentials in preventing oxidant-induced injury in the neonate lung.
Neonatal pulmonary oxidative stress was obvious after hyperoxia exposure regardless of genotypes, while Nrf2
deficiency elevated oxidative proteins and lipid peroxidation at baseline as well as after hyperoxia. Although widely used, the amount of MDA as a lipid peroxidation marker is known to be affected by several variables (22
). It would be worth validating the effect of Nrf2
deletion on oxidant tissue injury by measurement of a more reliable lipid peroxidation marker 8-iso
-Prostaglandin F2α (32
). Importantly, oxidative DNA damage is considered a causative factor in diverse pulmonary disorders, including neoplasia and acute lung injury. Previous studies have shown that hyperoxia caused base adduct formation (e.g.
, 7,8-dihydro-8-oxo-guanine) and DNA strand breakage in lungs of adult mice (4
); DNA adduct formation was found in most lung cells after the exposure, while the most severe level of damage, phosphodiester backbone breakage, was found only in type 2 cells, which resulted in the cell death and was associated with lung injury. Consistent with Nrf2-dependent variation in pulmonary apoptotic cell death and DNA lesions, microarray analysis also identified Nrf2-dependent dysregulation of genes involved in the DNA damage/repair and methylation pathways under conditions of normoxia (e.g.
) and hyperoxia (e.g.
). In particular, significant suppression of mitochondrial superoxide dismutase (Sod2
; Supplementary Table S4a
) is likely to be a factor leading to increased mitochondrial damage in Nrf2−/−
neonates. Although epigenetic effects of hyperoxia and Nrf2
dependency were beyond the scope of the current analysis, evidence indicates that hyperoxia causes hypermethylation in CpG islands of a lung gene in rats (50
). Further, hyperoxia-induced DNA damage influenced global DNA methylation status in lung epithelial cells (35
Impaired pulmonary vasculature development in ventilated preterm infants is thought to be caused by complex interactions of lung immaturity and postnatal factors including O2
, which results in arrest of alveolar growth (42
). Maturation of pulmonary vessel walls in BPD involves numerous growth components, including VEGF-A and ANGPTL2, and other factors, such as angiogenins and extracellular matrix proteins (42
). In the current study, lung ANGPTL2 proteins and Ang3
transcripts were suppressed in Nrf2−/−
mice relative to Nrf2+/+
mice constitutively and/or after hyperoxia, and 5′-flanking regions of Ang2
contained potential AREs. We speculate that although there was no effect on normal lung maturation, the differential constitutive levels of these proteins between two genotypes may predispose Nrf2−/−
neonates to enhanced susceptibility to hyperoxia. Other multiple angiogenic or antiangiogenic genes (e.g.
, and Itga1
) were also Nrf2 dependent after hyperoxia exposure. Overall, observations imply a potential adverse effect of Nrf2
deficiency on vessel development and endothelial differentiation in the saccular lung.
In conclusion, Nrf2 modulates genes involved in sustaining lung morphogenesis, cell growth machinery, and lymphocyte immunity during saccular lung maturation (). Nrf2 is also critical to protection of immature lungs from development of oxidative stress phenotypes caused by hyperoxia. Genetic loss of Nrf2 caused augmented oxidation, inflammation, DNA lesions, and aberrant alveolarization of saccular lungs (). Transcriptome analysis suggested that Nrf2 in the immature lung protected against O2 toxicity through regulation of DNA replication and cell cycle, various metabolism and small molecular process, and cell–cell interaction as well as redox homeostasis (). Functional bioinformatics elucidated downstream targets and in vivo validation of the role for Gpx2 and Marco indicated that Nrf2 has a dual role in lung maturation and protection against hyperoxia-induced lung injury. Results contribute to our understanding of the role of Nrf2 in molecular processes of alveolarization and in lung diseases of prematurity, and may suggest a potential therapeutic role for specific Nrf2 inducers (agonists) in protection against human BPD.
FIG. 7. Hypothetical schematics depicting proposed role for pulmonary Nrf2 in saccular-to-alveolar transition and hyperoxia-induced lung injury pathogenesis learned from mice. During early postnatal lung maturation and development, Nrf2 modulates expression of (more ...)