[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · May 2012 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2012 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: To assess incidence, burden of illness, and risk factors for human rhinoviruses (HRVs) in a cohort of very low birth weight (VLBW) infants.
A 2-year prospective cohort study was conducted among VLBW premature infants in Buenos Aires, Argentina. Infants were enrolled in the NICU from June 1, 2003, to May 31, 2005, and managed monthly and with every acute respiratory illness (ARI) during the first year of life. Nasal wash samples were obtained during every respiratory episode and tested for HRV, respiratory syncytial virus (RSV), human parainfluenza viruses, influenza viruses, and human metapneumovirus using reverse transcriptase-polymerase chain reaction.
Of 119 patients, 66 (55%) had HRV-associated ARIs. The incidence of HRV-associated ARI was 123 events per 100 child-years of follow-up. Of those infants experiencing an episode of bronchiolitis, 40% had HRV versus 7% with RSV. The incidence of HRV-associated bronchiolitis was 75 per 100 infant-years of follow-up. HRV was associated with 12 of 36 hospitalizations (33%), and RSV was associated with 9 of 36 hospitalizations (25%). The incidence of HRV-associated hospitalization was 12 per 100 infant-years of follow-up. The risk of HRV-associated hospitalization was higher for infants with bronchopulmonary dysplasia and those who were not breastfed.
HRV is an important and frequent pathogen associated with severe respiratory infections in VLBW infants. Bronchopulmonary dysplasia and the absence of breastfeeding are risk factors for hospitalization. The results of our study reveal that HRV is the predominant pathogen of respiratory infections in premature infants.
[Show abstract][Hide abstract] ABSTRACT: Airway cytotoxicity and secretion in pre-neoplastic stage. Giemsa-stained cytocentrifuge slides of bronchoalveolar lavage (BAL) fluid show increased BAL cell populations and greater cell clustering and lysis caused by urethane in Nrf2-/- than in Nrf2+/+ mice before tumor development at 9 wk (inset: higher magnification of BAL cells). Enhanced airway secretion (black arrow heads) and epithelial mucous production (black arrows) as indicated by AB/PAS-stained bronchial airway sections at 9 wk was consistent with clustering of the BAL cells in Nrf2-/- mice (inset: lower magnification of proximal airway and airspace). At 11 wk, clustering was resolved but greater cell lysis and cell debris in Nrf2-/- mice were accompanied by appearance of macrophages with phagocytosis-like features (red arrows) Insets present higher magnification of phagocytic macrophages. Representative slides showing intermediate magnitude of pathology for each treatment group are presented. AV, alveoli; BR, bronchi; PA, pulmonary artery. Bars indicate 100 µm.
[Show abstract][Hide abstract] ABSTRACT: Confirmation of microarray transcript profiles. (A) Expression of selected antioxidant/defense gene transcripts that were found by microarray analysis to be significantly increased or decreased by urethane in tumor tissues (22 wk): aldo-keto reductase family 1, member B8 (Akr1b8), aldehyde oxidase 3 (Aox3), peroxiredoxin 1 (Prdx1), glutathione peroxidase 2 (Gpx2), glutathione-S-transferase-µ (Gstm), and aldehyde dehydrogenase 1 family, member A1 (Aldh1a1). Relative suppression of these cytoprotective genes was evident in Nrf2-/- mice. Total glutathione (GSH) was determined in whole lung homogenates using a colorimetric kinetic analysis to support the differential induction of key GSH synthesis enzymes (e.g., glutathione synthetase, Gss; glutamate-cystein ligase, catalytic subunit, Gclc; glucose-6-phosphate dehydrogenase X-linked, G6pdx) between Nrf2+/+ and Nrf2-/- mice during the tumorigenesis. Mean±SEM are presented (n = 3/group). *, p<0.05 vs. genotype-matched saline control mice. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. Expression of selected lung genes significantly varied between Nrf2+/+ and Nrf2-/- mice at 12 wk (B) and at 22 wk (C) were determined by qRT-PCR. All qRT-PCR graphs depicted fold differences relative to Nrf2+/+ saline level of 18s-normalized data. All qRT-PCR data are represented as group mean±SEM (n = 3/group).
[Show abstract][Hide abstract] ABSTRACT: Top functional networks of Nrf2-dependent genes changed during urethane-induced tumorigenesis. (A) Ingenuity Pathway Analysis (IPA) generated a key network (Score 54) of cell-to-cell signaling and interaction-connective tissue development and function-ophthalmic disease with Nrf2-dependently regulated genes in pre-/early-neoplastic microenvironment (12 wk), in which genes encoding matrix metalloproteinase 2 (Mmp2) and D site albumin promoter binding protein (Dbp) were mapped as core molecules. (B) Nrf2-dependent lung tumor genes were highly associated in the networks of cell cycle-cancer-connective tissue development and function (Score 43, e.g., cyclin D1, Ccnd1; E2F transcription factor 3, E2f3), cell-to-cell signaling and interaction-tissue development-cell function (Score 38, e.g., chemokine (C-X-C motif) ligand 1, Cxcl1; integrin alpha 4, Itga4), and tumor morphology-cancer-dermatological disease and conditions (Score 38, e.g., G protein-coupled 56, Gpr56; glutathione-S-transferase, alpha 3, Gsta3) as generated by IPA. (C) Nrf2-dependently modulated genes in common (n = 21 genes) in early-neoplastic microenvironment (12 wk) and in lung tumors (22 wk) were functionally associated in cancer-cell cycle-cell death network (Score 23). They included genes encoding CD34, UGT1a1, fetuin beta (Fetub), G protein-coupled receptor 137B (Gpr137b), ATP binding cassette, subfamily C, member 4 (Abcc4), phosphogluconate dehydrogenase (Pgd), etc. (.ppt).
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Top functional networks of significantly changed gene transcripts in lung tumors of Nrf2+/+ mice at 22 wk. Ingenuity Pathway Analysis (IPA) generated essential functional networks of the genes significantly (≥2-fold, n = 3461) changed in Nrf2+/+ tumors. Highest association score (40) was for the genetic disorder-skeletal and muscular disorders- developmental disorder network (A) where the genes such as coiled-coil domain containing 85A, (Ccdc85a), dystrophin, muscular dystrophy (Dmd), and fyn proto-oncogene (Fyn) were mapped. Many genes (e.g., CCAAT/enhancer binding protein (C/EBP) alpha, Cebpa; lymphotoxin B, Ltb) were identified for cellular development-cellular growth and proliferation-hematological system development and function-hematopoiesis networks (B and C, Scores 34 and 33, respectively) and others related in cell cycle- cellular movement-cancer network (D, score 33, e.g., p21, Cdkn1a; cell division cycle 20, Cdc20) or in amino acid metabolism-molecular transport-small molecular biochemistry (E, Score 33, e.g., platelet derived growth factor receptor, beta polypeptide Pdgfrb; platelet-activating factor acetylhydrolase, isoform 1b, subunit 3, Pafah1b3) were also closely associated during tumorigenesis. (.ppt).