High-density mapping of mammalian genomes has enabled a wide range of genetic investigations including the mapping of polygenic traits, determination of quantitative trait loci, and phylogenetic comparison. Genome sequencing analysis of inbred mouse strains has identified high-density single nucleotide polymorphisms (SNPs) for investigation of complex traits, which has become a useful tool for biomedical research of human disease to alleviate ethical and practical problems of experimentation in humans. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) encodes a key host defense transcription factor. This review describes genetic characteristics of human NRF2 and its homologs in other vertebrate species. NRF2 is evolutionally conserved and shares sequence homology among species. Compilation of publically available SNPs and other genetic mutations shows that human NRF2 is highly polymorphic with a mutagenic frequency of 1 per every 72 bp. Functional at-risk alleles and haplotypes have been demonstrated in various human disorders. In addition, other pathogenic alterations including somatic mutations and misregulated epigenetic processes in NRF2 have led to oncogenic cell survival. Comprehensive information from the current review addresses association of NRF2 variation and disease phenotypes and supports the new insights into therapeutic strategies.
Ozone (O3) is a strong oxidant in air pollution that has harmful effects on airways and exacerbates respiratory disorders. The transcription factor Nrf2 protects airways from oxidative stress through antioxidant response element-bearing defense gene induction. The present study was designed to determine the role of Nrf2 in airway toxicity caused by inhaled O3 in mice. For this purpose, Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice received acute and subacute exposures to O3. Lung injury was determined by bronchoalveolar lavage and histopathologic analyses. Oxidation markers and mucus hypersecretion were determined by ELISA, and Nrf2 and its downstream effectors were determined by RT-PCR and/or Western blotting. Acute and sub-acute O3 exposures heightened pulmonary inflammation, edema, and cell death more severely in Nrf2−/− mice than in Nrf2+/+ mice. O3 caused bronchiolar and terminal bronchiolar proliferation in both genotypes of mice, while the intensity of compensatory epithelial proliferation, bronchial mucous cell hyperplasia, and mucus hypersecretion was greater in Nrf2−/− mice than in Nrf2+/+ mice. Relative to Nrf2+/+, O3 augmented lung protein and lipid oxidation more highly in Nrf2−/− mice. Results suggest that Nrf2 deficiency exacerbates oxidative stress and airway injury caused by the environmental pollutant O3.
Aims: Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current 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. Results: 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 than 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. Innovation: 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. Conclusion:
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. Antioxid. Redox Signal. 00, 000–000.
Nrf2 protects the lung from adverse responses to oxidants, including 100% oxygen (hyperoxia) and airborne pollutants like particulate matter (PM) exposure, but 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.
Exposure of mice to hyperoxia produces pulmonary toxicity similar to acute lung injury/acute respiratory distress syndrome, but little is known about the interactions within the cardiopulmonary system. This study was designed to characterize the cardiopulmonary response to hyperoxia, and to identify candidate susceptibility genes in mice. Electrocardiogram and ventilatory data were recorded continuously from 4 inbred and 29 recombinant inbred strains during 96 hours of hyperoxia (100% oxygen). Genome-wide linkage analysis was performed in 27 recombinant inbred strains against response time indices (TIs) calculated from each cardiac phenotype. Reductions in minute ventilation, heart rate (HR), low-frequency (LF) HR variability (HRV), high-frequency HRV, and total power HRV were found in all mice during hyperoxia exposure, but the lag time before these changes began was strain dependent. Significant (chromosome 9) or suggestive (chromosomes 3 and 5) quantitative trait loci were identified for the HRTI and LFTI. Functional polymorphisms in several candidate susceptibility genes were identified within the quantitative trait loci and were associated with hyperoxia susceptibility. This is the first study to report highly significant interstrain variation in hyperoxia-induced changes in minute ventilation, HR, and HRV, and to identify polymorphisms in candidate susceptibility genes that associate with cardiac responses. Results indicate that changes in HR and LF HRV could be important predictors of subsequent adverse outcome during hyperoxia exposure, specifically the pathogenesis of acute lung injury. Understanding the genetic mechanisms of these responses may have significant diagnostic clinical value.
heart rate; hyperoxia; genome-wide mapping
Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2+/+ and Nrf2-/- mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2-/- mice compared to Nrf2+/+ mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2-/- mice than in Nrf2+/+ mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2+/+ mice relative to Nrf2-/- mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.
Ozone (O3) remains a prevalent air pollutant and public health concern. Inf2 is a significant quantitative trait locus (QTL) on murine chromosome 17 that contributes to susceptibility to O3-induced infiltration of polymorphonuclear leukocytes (PMNs) into the lung, but the mechanisms of susceptibility remain unclear. The study objectives were to confirm and restrict Inf2, and to identify and test novel candidate susceptibility gene(s).
Congenic strains of mice that contained overlapping regions of Inf2 and their controls, and mice deficient in either MHC Class II genes or the Tnf cluster were exposed to air or O3. Lung inflammation and gene expression were assessed.
Inf2 was restricted from 16.42 Mbp to 0.96 Mbp, and bioinformatic analysis identified MHC class II, the Tnf cluster, and other genes in this region that contain potentially informative SNPs between the susceptible and resistant mice. Furthermore, O3-induced inflammation was significantly reduced in mice deficient in MHC class II genes, or the Tnf cluster genes, compared to wild-type controls. Gene expression differences were also observed in MHC class II and Tnf cluster genes.
This integrative genetic analysis of Inf2 led to identification of novel O3 susceptibility genes that may provide important, new therapeutic targets in susceptible individuals.
inflammation; lymphotoxin α; mouse; major histocompatibility complex; susceptibility; tumor necrosis factor
Rationale: The NF-E2 related factor 2 (Nrf2)–antioxidant response element (ARE) pathway is essential for protection against oxidative injury and inflammation including hyperoxia-induced acute lung injury. Microarray expression profiling revealed that lung peroxisome proliferator activated receptor γ (PPARγ) induction is suppressed in hyperoxia-susceptible Nrf2-deficient (Nrf2−/−) mice compared with wild-type (Nrf2+/+) mice. PPARγ has pleiotropic beneficial effects including antiinflammation in multiple tissues.
Objectives: We tested the hypothesis that PPARγ is an important determinant of pulmonary responsivity to hyperoxia regulated by Nrf2.
Methods: A computational bioinformatic method was applied to screen potential AREs in the Pparg promoter for Nrf2 binding. The functional role of a potential ARE was investigated by in vitro promoter analysis. A role for PPARγ in hyperoxia-induced acute lung injury was determined by temporal silencing of PPARγ via intranasal delivery of PPARγ-specific interference RNA and by administration of a PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 in mice.
Measurements and Main Results: Deletion or site-directed mutagenesis of a potential ARE spanning -784/-764 sequence significantly attenuated hyperoxia-increased Pparg promoter activity in airway epithelial cells overexpressing Nrf2, indicating that the -784/-764 ARE is critical for Nrf2-regulated PPARγ expression. Mice with decreased lung PPARγ by specific interference RNA treatment had significantly augmented hyperoxia-induced pulmonary inflammation and injury. 15 Deoxy-Δ12,14-prostaglandin J2 administration significantly reduced hyperoxia-induced lung inflammation and edema in Nrf2+/+, but not in Nrf2−/− mice.
Conclusions: Results indicate for the first time that Nrf2-driven PPARγ induction has an essential protective role in pulmonary oxidant injury. Our observations provide new insights into the therapeutic potential of PPARγ in airway oxidative inflammatory disorders.
antioxidant response element; hyperoxia; inflammation; siRNA; 15d-PGJ2
The mechanisms underlying ozone (O3)-induced pulmonary inflammation remain unclear. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators.
We investigated the molecular mechanisms underlying interleuken-10 (IL-10)–mediated attenuation of O3-induced pulmonary inflammation in mice.
Il10-deficient (Il10−/−) and wild-type (Il10+/+) mice were exposed to 0.3 ppm O3 or filtered air for 24, 48, or 72 hr. Immediately after exposure, differential cell counts and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also used global mRNA expression analyses of lung tissue with Ingenuity Pathway Analysis to identify patterns of gene expression through which IL-10 modifies O3-induced inflammation.
Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10−/− mice than in Il10+/+ mice after exposure to O3 at all time points tested. O3-enhanced nuclear NF-κB translocation was elevated in the lungs of Il10−/− compared with Il10+/+ mice. Gene expression analyses revealed several IL-10–dependent and O3-dependent mediators, including macrophage inflammatory protein 2, cathepsin E, and serum amyloid A3.
Results indicate that IL-10 protects against O3-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several genetic targets through which IL-10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O3-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals.
air pollution; gene array; IL-10; inflammation; lung; ozone; pulmonary
Rationale: Respiratory syncytial virus (RSV) is the most frequent cause of significant lower respiratory illness in infants and young children, but its pathogenesis is not fully understood. The transcription factor Nrf2 protects lungs from oxidative injury and inflammation via antioxidant response element (ARE)-mediated gene induction.
Objectives: The current study was designed to determine the role of Nrf2-mediated cytoprotective mechanisms in murine airway RSV disease.
Methods: Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice were intranasally instilled with RSV or vehicle. In a separate study, Nrf2+/+ and Nrf2−/− mice were treated orally with sulforaphane (an Nrf2-ARE inducer) or phosphate-buffered saline before RSV infection.
Measurements and Main Results: RSV-induced bronchopulmonary inflammation, epithelial injury, and mucus cell metaplasia as well as nasal epithelial injury were significantly greater in Nrf2−/− mice than in Nrf2+/+ mice. Compared with Nrf2+/+ mice, significantly attenuated viral clearance and IFN-γ, body weight loss, heightened protein/lipid oxidation, and AP-1/NF-κB activity along with suppressed antioxidant induction was found in Nrf2−/− mice in response to RSV. Sulforaphane pretreatment significantly limited lung RSV replication and virus-induced inflammation in Nrf2+/+ but not in Nrf2−/− mice.
Conclusions: The results of this study support an association of oxidant stress with RSV pathogenesis and a key role for the Nrf2-ARE pathway in host defense against RSV.
airway; oxidative stress; antioxidant response element; inflammation; sulforaphane
Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes, and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system we: (1) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (2) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (3) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (4) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed; and (5) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.
Redox imbalance has been implicated in the pathogenesis of many acute and chronic lung diseases. The b-Zip transcription factor Nrf2 acts via an antioxidant/electrophilic response element to regulate antioxidants and maintain cellular redox homeostasis. Our previous studies have shown that Nrf2-deficient mice (Nrf2−/−) show reduced pulmonary expression of several antioxidant enzymes, which renders them highly susceptible to hyperoxia-induced lung injury. To better understand the physiologic significance of Nrf2-induced redox signaling, we have used primary cells isolated from the lungs of Nrf2+/+ and Nrf2−/− mice. Our studies were focused on type II cells because these cells are constantly exposed to the oxidant environment and play key roles in host defense, injury, and repair processes. Using this system, we now report that an Nrf2 deficiency leads to defects in type II cell proliferation and greatly enhances the cells' sensitivity to oxidant-induced cell death. These defects were closely associated with high levels of reactive oxygen species (ROS) and redox imbalance in Nrf2−/− cells. Glutathione (GSH) supplementation rescued these phenotypic defects associated with the Nrf2 deficiency. Intriguingly, although the antioxidant N-acetyl-cysteine drastically squelched ROS levels, it was unable to counteract growth arrest in Nrf2−/− cells. Moreover, despite their elevated levels of ROS, Nrf2−/− type II cells were viable and, like their wild-type counterparts, exhibited normal differentiation characteristics. Our data suggest that dysfunctional Nrf2-regulated GSH-induced signaling is associated with deregulation of type II cell proliferation, which contributes to abnormal injury and repair and leads to respiratory impairment.
oxidative stress; lung; antioxidants; cell proliferation
Rationale: Increasing evidence suggests that tumor necrosis factor (TNF)-α plays a key role in pulmonary injury caused by environmental ozone (O3) in animal models and human subjects. We previously determined that mice genetically deficient in TNF response are protected from lung inflammation and epithelial injury after O3 exposure.
Objectives: The present study was designed to determine the molecular mechanisms of TNF receptor (TNF-R)–mediated lung injury induced by O3.
Methods: TNF-R knockout (Tnfr−/−) and wild-type (Tnfr+/+) mice were exposed to 0.3 ppm O3 or air (for 6, 24, or 48 h), and lung RNA and proteins were prepared. Mice deficient in p50 nuclear factor (NF)-κB (Nfkb1−/−) or c-Jun–NH2 terminal kinase 1 (Jnk1−/−) and wild-type controls (Nfkb1+/+, Jnk1+/+) were exposed to O3 (48 h), and the role of NF-κB and mitogen-activated protein kinase (MAPK) as downstream effectors of lung injury was analyzed by bronchoalveolar lavage analyses.
Results: O3-induced early activation of TNF-R adaptor complex formation was attenuated in Tnfr−/− mice compared with Tnfr+/+ mice. O3 significantly activated lung NF-κB in Tnfr+/+ mice before the development of lung injury. Basal and O3-induced NF-κB activity was suppressed in Tnfr−/− mice. Compared with Tnfr+/+ mice, MAPKs and activator protein (AP)-1 were lower in Tnfr−/− mice basally and after O3. Furthermore, inflammatory cytokines, including macrophage inflammatory protein-2, were differentially expressed in Tnfr−/− and Tnfr+/+ mice after O3. O3-induced lung injury was significantly reduced in Nfkb1−/− and Jnk1−/− mice relative to respective control animals.
Conclusions: Results suggest that NF-κB and MAPK/AP-1 signaling pathways are essential in TNF-R–mediated pulmonary toxicity induced by O3.
tumor necrosis factor receptor; knockout; nuclear factor-κB; mitogen-activated protein kinase; activator protein-1
Exposure to ozone causes airway inflammation, hyperreactivity, lung hyper-permeability, and epithelial cell injury. An early inflammatory response induced by inhaled O3 is characterized primarily by release of inflammatory mediators such as cytokines, chemokines, and airway neutrophil accumulation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of oxidative lung disorders including acute lung injury, asthma, and chronic obstructive pulmonary disease.
We hypothesized that MMPs have an important role in the pathogenesis of O3-induced airway inflammation.
We compared the lung injury responses in either Mmp7- (Mmp7−/−) or Mmp9-deficient (Mmp9−/−) mice and their wild-type controls (Mmp7+/+, Mmp9+/+) after exposure to 0.3 ppm O3 or filtered air.
Relative to air-exposed controls, MMP-9 activity in bronchoalveolar lavage fluid (BALF) was significantly increased by O3 exposure in Mmp9+/+ mice. O3-induced increases in the concentration of total protein (a marker of lung permeability) and the numbers of neutrophils and epithelial cells in BALF were significantly greater in Mmp9−/− mice compared with Mmp9+/+ mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 levels in BALF were also significantly higher in Mmp9−/− mice than in Mmp9+/+ mice after O3 exposure, although no differences in mRNA expression for these chemokines were found between genotypes. Mean BALF protein concentration and numbers of inflammatory cells were not significantly different between Mmp7+/+ and Mmp7−/− mice after O3 exposure.
Results demonstrated a protective role of MMP-9 but not of MMP-7, in O3-induced lung neutrophilic inflammation and hyperpermeability. The mechanism through which Mmp9 limits O3-induced airway injury is not known but may be via posttranscriptional effects on proinflammatory CXC chemokines including KC and MIP-2.
chemokine; knockout mice; lung; MMP-9; O3; oxidant