Preconditioning the immature lung with enhanced Nrf2 activity protects against oxidant-induced hypoalveolarization in mice.

Bronchopulmonary dysplasia (BPD) is a chronic disease of preterm babies with poor clinical outcomes. Nrf2 transcription factor is crucial for cytoprotective response, whereas Keap1-an endogenous inhibitor of Nrf2 signaling-dampens these protective responses. Nrf2-sufficient (wild type) newborn mice exposed to hyperoxia develop hypoalveolarization, which phenocopies human BPD, and Nrf2 deficiency worsens it. In this study, we used PND1 pups bearing bearing hypomorphic Keap1 floxed alleles (Keap1f/f) with increased levels of Nrf2 to test the hypothesis that constitutive levels of Nrf2 in the premature lung are insufficient to mitigate hyperoxia-induced hypoalveolarization. Both wildtype and Keap1f/f pups at PND1 were exposed to hyperoxia for 72 h and then allowed to recover at room air for two weeks (at PND18), sacrificed, and lung hypoalveolarization and inflammation assessed. Hyperoxia-induced lung hypoalveolarization was remarkably lower in Keap1f/f pups than in wildtype counterparts (28.9% vs 2.4%, wildtype vs Keap1f/f). Likewise, Keap1f/f pups were protected against prolonged (96 h) hyperoxia-induced hypoalveolarization. However, there were no differences in hyperoxia-induced lung inflammatory response immediately after exposure or at PND18. Lack of hypoalveolarization in Keap1f/f pups was accompanied by increased levels of expression of antioxidant genes and GSH as assessed immediately following hyperoxia. Keap1 knockdown resulted in upregulation of lung cell proliferation postnatally but had opposing effects following hyperoxia. Collectively, our study demonstrates that augmenting endogenous Nrf2 activation by targeting Keap1 may provide a physiological way to prevent hypoalveolarization associated with prematurity.

FOSL1 Promotes Kras-induced Lung Cancer through Amphiregulin and Cell Survival Gene Regulation.

The FOSL1/AP-1 transcription factor regulates gene expression, thereby controlling various pathophysiological processes. It is a major effector of RAS-ERK1/2 signaling and is activated in human lung epithelia by tumorigenic stimuli. Recent evidence shows an inverse correlation between FOSL1 expression and the survival of patients with lung cancer and adenocarcinomas; however, its role in lung tumorigenesis remains elusive. In this work, we sought to determine the role of FOSL1 in Kras-induced lung adenocarcinoma in vivo and its downstream effector mechanisms. We used mice expressing the Kras oncogene in the lung with concomitant Fosl1 deletion, Kras-activated murine alveolar epithelial cells (mAECs) with Fosl1 deletion, and KRAS mutant human lung adenocarcinoma (HLAC) cells with FOSL1 deficiency, and performed cell proliferation and gene expression analyses. Mutant Kras induced Fosl1 expression in vitro (mAECs) and in vivo (lung tissue), and mice with Fosl1 deletion showed reduced levels of mutant Kras-induced lung tumorigenesis and survived longer than Fosl1-sufficient mice. Studies with mutant Kras-activated mAECs and KRAS-mutant HLAC cells revealed that FOSL1 regulates mutant KRAS-induced gene expression, thereby controlling cell proliferation and survival. In contrast, FOSL1 depletion in non-KRAS-mutant HLAC cells and nonmalignant human lung epithelia had no effect. Our data support the notion that FOSL1-mediated expression of amphiregulin and apoptotic and antioxidative genes plays a role in regulating HLAC cell proliferation and survival. FOSL1 is a determinant of lung cancer in vivo and regulates HLAC cell proliferation and survival, largely in the context of KRAS mutations. Activation of FOSL1 in adenocarcinomas may be a prognostic marker and potential target for human lung cancer with KRAS mutations.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia.

BACKGROUND: Sphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1 -/- ) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1 -/- mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1 -/- neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45. CONCLUSION: Using SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

K-homology splicing regulatory protein (KSRP) promotes post-transcriptional destabilization of Spry4 transcripts in non-small cell lung cancer.

AU-rich element-binding proteins (ARE-BPs) offer post-transcriptional regulation of gene expression via physical interaction and recruitment of RNA decay machinery to the AU-rich elements within the 3'-UTR of the target transcripts. However, the role of ARE-BPs in lung cancer remains poorly understood. In this study, we have identified that K-homology splicing regulatory protein (KSRP), an ARE-BP, is robustly up-regulated in human lung cancer. Importantly, Kaplan-Meier survival analysis indicated that elevated KSRP expression was correlated with poor overall survival of lung cancer patients. Furthermore, cigarette smoke, a leading risk factor for lung cancer, was also identified to be an important contributor to increased KSRP expression. Remarkably, silencing of KSRP decreased cell proliferation, reversed anchorage-independent growth, and reduced migration/invasion, suggesting an oncogenic role for KSRP in lung cancer. Finally, we provide mechanistic evidence that KSRP promotes the down-regulation of Spry4 by a previously unidentified mechanism, i.e. post-transcriptional mRNA regulation.

Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation.

Ischemia-reperfusion (IR)-induced kidney injury is a major clinical problem, but its underlying mechanisms remain unclear. The transcription factor known as nuclear factor, erythroid 2-like 2 (NFE2L2 or Nrf2) is crucial for protection against oxidative stress generated by pro-oxidant insults. We have previously shown that Nrf2 deficiency enhances susceptibility to IR-induced kidney injury in mice and that its upregulation is protective. Here, we examined Nrf2 target antioxidant gene expression and the mechanisms of its activation in both human and murine kidney epithelia following acute (2 h) and chronic (12 h) hypoxia and reoxygenation conditions. We found that acute hypoxia modestly stimulates and chronic hypoxia strongly stimulates Nrf2 putative target HMOX1 expression, but not that of other antioxidant genes. Inhibition of AKT1/2 or ERK1/2 signaling blocked this induction; AKT1/2 but not ERK1/2 inhibition affected Nrf2 levels in basal and acute hypoxia-reoxygenation states. Unexpectedly, chromatin immunoprecipitation assays revealed reduced levels of Nrf2 binding at the distal AB1 and SX2 enhancers and proximal promoter of HMOX1 in acute hypoxia, accompanied by diminished levels of nuclear Nrf2. In contrast, Nrf2 binding at the AB1 and SX2 enhancers significantly but differentially increased during chronic hypoxia and reoxygenation, with reaccumulation of nuclear Nrf2 levels. Small interfering-RNA-mediated Nrf2 depletion attenuated acute and chronic hypoxia-inducible HMOX1 expression, and primary Nrf2-null kidney epithelia showed reduced levels of HMOX1 induction in response to both acute and chronic hypoxia. Collectively, our data demonstrate that Nrf2 upregulates HMOX1 expression in kidney epithelia through a distinct mechanism during acute and chronic hypoxia reoxygenation, and that both AKT1/2 and ERK1/2 signaling are required for this process.

Sirtuin 1 Promotes Hyperoxia-Induced Lung Epithelial Cell Death Independent of NF-E2-Related Factor 2 Activation.

Lung epithelial cell damage accompanied by death is a cardinal feature of toxicant- and prooxidant-induced acute lung injury. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NEF2L2 or NRF2) activates several antioxidant enzymes (AOEs) and prosurvival genes in response to oxidant stress, and its deficiency enhances susceptibility to hyperoxic lung injury and other oxidant-induced lung pathologies. Sirtuin 1 (SIRT1) regulates cell growth and survival in response to both physiological and pathological stresses by selectively deacetylating multiple proteins required for chromatin remodeling and transcription; therefore, we sought to examine potential SIRT1-NRF2 cross-talk in the regulation of AOE expression during hyperoxia-induced lung epithelial cell death. Unexpectedly, pharmacological inhibition or small interfering RNA-mediated depletion of SIRT1 caused a reduction in cell death, accompanied by reduced levels of NRF2-dependent AOE expression in chronic hyperoxia. NRF2 acetylation was markedly and transiently higher in cells exposed to acute (6 h) hyperoxia. Sirtinol blocked this acute effect, but NRF2 acetylation was low or undetectable in cells exposed to chronic hyperoxia (24-36 h) both with and without sirtinol. SIRT1 activation by resveratrol augmented hyperoxia-induced death in cells with NRF2 deficiency. SIRT1 inhibition or depletion led to a reduced activation of the cell-death executioner caspase 3, whereas caspase inhibition prevented death. Consistent with these results, sirtinol attenuated hyperoxia-induced lung alveolar permeability and toxicity in vivo. Collectively, these results reveal that, in chronic hyperoxia, SIRT1 promotes hyperoxia-induced lung epithelial cell damage and death by altering pro- and antiapoptotic balance, not by dampening optimal NRF2-dependent AOE expression.

Visualization of Fra-1/AP-1 activation during LPS-induced inflammatory lung injury using fluorescence optical imaging.

Inappropriate lung inflammatory response following oxidant and toxicant exposure can lead to abnormal repair and disease pathogenesis, including fibrosis. Thus early detection of molecular and cellular processes and mediators promoting lung inflammation is necessary to develop better strategies for therapeutic intervention and disease management. Previously, we have shown that transcription factor Fra-1/AP-1 plays key roles in lung inflammatory response, as Fra-1-null mice are less susceptible than wild-type mice to LPS-induced lung injury and mortality. Herein, we developed a transgenic reporter mouse model expressing tdTomato under the control of FRA-1 (human) promoter (referred to as FRA-1(TdTg) mice) to monitor its activation during inflammatory lung injury using fluorescence protein-based optical imaging and molecular analysis in vivo and ex vivo. A higher red fluorescent signal was observed in the lungs of LPS-treated FRA-1(TdTg) mice compared with vehicle controls, and Western blot and qRT-PCR analyses revealed a significant correlation with the FRA-1-tdTomato reporter expression. Immunocolocalization demonstrated expression of FRA-1-tdTomato largely in lung alveolar macrophages and to some extent in epithelial cells. Moreover, we validated these results with a second reporter mouse model that expressed green fluorescent protein upon activation of endogenous Fra-1 promoter. Additionally, we demonstrated increased expression of FRA-1 in alveolar macrophages in human lung instilled with Escherichia coli ex vivo. Collectively, our data obtained from two independent reporter mouse models and from human samples underscore the significance of Fra-1 activation in alveolar macrophages during inflammatory lung injury and may aid in developing strategies to target this transcription factor in lung injury and repair.

PI3K-AKT Signaling via Nrf2 Protects against Hyperoxia-Induced Acute Lung Injury, but Promotes Inflammation Post-Injury Independent of Nrf2 in Mice.

Lung epithelial and endothelial cell death accompanied by inflammation contributes to hyperoxia-induced acute lung injury (ALI). Impaired resolution of ALI can promote and/or perpetuate lung pathogenesis, including fibrosis. Previously, we have shown that the transcription factor Nrf2 induces cytoprotective gene expression and confers protection against hyperoxic lung injury, and that Nrf2-mediated signaling is also crucial for the restoration of lung homeostasis post-injury. Although we have reported that PI3K/AKT signaling is required for Nrf2 activation in lung epithelial cells, significance of the PI3K/AKT-Nrf2 crosstalk during hyperoxic lung injury and repair remains unclear. Thus, we evaluated this aspect using Nrf2 knockout (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mouse models. Here, we show that pharmacologic inhibition of PI3K/AKT signaling increased lung inflammation and alveolar permeability in Nrf2(+/+) mice, accompanied by decreased expression of Nrf2-target genes such as Nqo1 and Hmox1. PI3K/AKT inhibition dampened hyperoxia-stimulated Nqo1 and Hmox1 expression in lung epithelial cells and alveolar macrophages. Contrasting with its protective effects, PI3K/AKT inhibition suppressed lung inflammation in Nrf2(+/+) mice during post-injury. In Nrf2(-/-) mice exposed to room-air, PI3K/AKT inhibition caused lung injury and inflammation, but it did not exaggerate hyperoxia-induced ALI. During post-injury, PI3K/AKT inhibition did not augment, but rather attenuated, lung inflammation in Nrf2(-/-) mice. These results suggest that PI3K/AKT-Nrf2 signaling is required to dampen hyperoxia-induced lung injury and inflammation. Paradoxically, the PI3K/AKT pathway promotes lung inflammation, independent of Nrf2, during post-injury.

The Nrf2 triterpenoid activator, CDDO-imidazolide, protects kidneys from ischemia-reperfusion injury in mice.

Acute kidney injury (AKI) caused by ischemia-reperfusion is a major clinical problem in both native and transplanted kidneys. We had previously shown that deficiency of Nrf2, a potent bZIP transcription factor that binds to the antioxidant response element, enhances susceptibility to experimental ischemic AKI. Here we further explored the role of Nrf2 in AKI by amplifying Nrf2 activation in vivo and in vitro with the synthetic triterpenoid CDDO-imidazolide. Mice treated with CDDO-imidazolide and undergoing experimental bilateral ischemic AKI had improved survival and renal function. Treated mice had improved renal histology with a decrease in tubular injury, as well as a decrease in proinflammatory cytokine and chemokine production compared with vehicle-treated mice. In an exploration of protective mechanisms, we found an upregulation of Nrf2 target antioxidant genes in CDDO-imidazolide-treated mouse kidneys. Furthermore, Nrf2-deficient mice treated with CDDO-imidazolide had no significant improvement in mortality, renal function or histology, proinflammatory cytokine gene expression, and no significant increase in antioxidant gene expression. In vitro studies demonstrated that the renal epithelial cells were likely an important target of CDDO-imidazolide. Thus, activation of Nrf2 signaling with CDDO-imidazolide confers protection from AKI, and presents a new therapeutic opportunity for this common and serious condition.

Sphingosine kinase 1 deficiency confers protection against hyperoxia-induced bronchopulmonary dysplasia in a murine model: role of S1P signaling and Nox proteins.

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

Expression profiling of genes regulated by Fra-1/AP-1 transcription factor during bleomycin-induced pulmonary fibrosis.

BACKGROUND: The Fra-1/AP-1 transcription factor regulates the expression of genes controlling various processes including migration, invasion, and survival as well as extracellular remodeling. We recently demonstrated that loss of Fra-1 leads to exacerbated bleomycin-induced pulmonary fibrosis, accompanied by enhanced expression of various inflammatory and fibrotic genes. To better understand the molecular mechanisms by which Fra-1 confers protection during bleomycin-induced lung injury, genome-wide mRNA expression profiling was performed. RESULTS: We found that Fra-1 regulates gene expression programs that include: 1) several cytokines and chemokines involved in inflammation, 2) several genes involved in the extracellular remodeling and cell adhesion, and 3) several genes involved in programmed cell death. CONCLUSION: Loss of Fra-1 leads to the enhanced expression of genes regulating inflammation and immune responses and decreased the expression of genes involved in apoptosis, suggesting that this transcription factor distinctly modulates early pro-fibrotic cellular responses.

The NRF2 activation and antioxidative response are not impaired overall during hyperoxia-induced lung epithelial cell death.

Lung epithelial and endothelial cell death caused by pro-oxidant insults is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients. The NF-E2-related factor 2 (NRF2) activation in response to oxidant exposure is crucial to the induction of several antioxidative and cytoprotective enzymes that mitigate cellular stress. Since prolonged exposure to hyperoxia causes cell death, we hypothesized that chronic hyperoxia impairs NRF2 activation, resulting in cell death. To test this hypothesis, we exposed nonmalignant small airway epithelial cells (AECs) to acute (1-12 h) and chronic (36-48 h) hyperoxia and evaluated cell death, NRF2 nuclear accumulation and target gene expression, and NRF2 recruitment to the endogenous HMOX1 and NQO1 promoters. As expected, hyperoxia gradually induced death in AECs, noticeably and significantly by 36 h; ~60% of cells were dead by 48 h. However, we unexpectedly found increased expression levels of NRF2-regulated antioxidative genes and nuclear NRF2 in AECs exposed to chronic hyperoxia as compared to acute hyperoxia. Chromatin Immunoprecipitation (ChIP) assays revealed an increased recruitment of NRF2 to the endogenous HMOX1 and NQO1 promoters in AECs exposed to acute or chronic hyperoxia. Thus, our findings demonstrate that NRF2 activation and antioxidant gene expression are functional during hyperoxia-induced lung epithelial cell death and that chronic hyperoxia does not impair NRF2 signaling overall.

Role of migratory inhibition factor in age-related susceptibility to radiation lung injury via NF-E2-related factor-2 and antioxidant regulation.

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.

Targeted deletion of Jun/AP-1 in alveolar epithelial cells causes progressive emphysema and worsens cigarette smoke-induced lung inflammation.

Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun-mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.

Genetic disruption of Fra-1 decreases susceptibility to endotoxin-induced acute lung injury and mortality in mice.

The activator protein-1 (AP-1) transcription factor, comprising Jun and Fos family proteins, distinctly regulates various cellular processes, including those involved in inflammation. FOS like antigen 1 (Fra-1), a member of the Fos family, dimerizes with members of the Jun family and regulates gene expression in a context-dependent manner. Although respiratory toxicants are known to stimulate the expression of Fra-1 in the lung, whether Fra-1 promotes or decreases susceptibility to the development and progression of toxicant-induced lung disease in vivo is not well established. To determine the role of Fra-1 in LPS-induced acute lung injury and mortality, we administered LPS either intraperitoneally or intratracheally to Fra-1-sufficient (Fra-11(+/+)) and Fra-1-deficient (Fra-1(Δ/Δ)) mice. LPS-induced mortality, lung injury, inflammation, cytokine measurements, and AP-1 and NF-κB activities were then assessed in these mice. Fra-1(Δ/Δ) mice showed a greater resistance to LPS-induced mortality than did their Fra-1(+/+) counterparts. Consistent with this result, LPS-induced lung injury and inflammatory responses were markedly lower in Fra-1(Δ/Δ) mice than in Fra-1(+/+) mice. Compared with Fra-1(+/+) mice, Fra-1(Δ/Δ) mice showed a reduced influx of neutrophils into the lungs, accompanied by a decreased expression of proinflammatory cytokines in response to treatment with LPS. The decreased inflammatory responses in Fra-1(Δ/Δ) mice coincided with diminished and increased levels of NF-κB and c-Jun/AP-1 binding, respectively. These results demonstrate that Fra-1/AP-1 plays a key role in promoting LPS-induced injury and mortality in mice, and they suggest that targeting (i.e., inhibiting) this transcription factor may be a useful approach to dampening the adverse effects of exposure to endotoxins.

Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice.

Aberrant tissue repair and persistent inflammation following oxidant-mediated acute lung injury (ALI) can lead to the development and progression of various pulmonary diseases, but the mechanisms underlying these processes remain unclear. Hyperoxia is widely used in the treatment of pulmonary diseases, but the effects of this oxidant exposure in patients undergoing recovery from ALI are not clearly understood. Nrf2 has emerged as a crucial transcription factor that regulates oxidant stress through the induction of several detoxifying enzymes and other proteins. Using an experimental model of hyperoxia-induced ALI, we have examined the role of oxidant stress in resolving lung injury and inflammation. We found that when exposed to sublethal (72 h) hyperoxia, Nrf2-deficient, but not wild-type mice, succumbed to death during recovery. When both genotypes were exposed to a shorter period of hyperoxia-induced ALI (48 h), the lungs of Nrf2-deficient mice during recovery exhibited persistent cellular injury, impaired alveolar and endothelial cell regeneration, and persistent cellular infiltration by macrophages and lymphocytes. Glutathione (GSH) supplementation in Nrf2-deficient mice immediately after hyperoxia remarkably restored their ability to recover from hyperoxia-induced damage in a manner similar to that of wild-type mice. Thus, the results of the present study indicate that the Nrf2-regulated transcriptional response and, particularly GSH synthesis, is critical for lung tissue repair and the resolution of inflammation in vivo and suggests that a dysfunctional Nrf2-GSH pathway may compromise these processes in vivo.

Epidermal growth factor receptor (EGFR) regulates mechanical ventilation-induced lung injury in mice.

Mechanical ventilation (MV) is used as therapy to support critically ill patients; however, the mechanisms by which MV induces lung injury and inflammation remain unclear. Epidermal growth factor receptor (EGFR)-mediated signaling plays a key role in various physiologic and pathologic processes, which include those modulated by mechanical and shear forces, in various cell types. We hypothesized that EGFR-activated signaling plays a key role in ventilator-induced lung injury and inflammation (VILI). To test this hypothesis, we assessed lung vascular and alveolar permeability as well as inflammation, which are cardinal features of VILI, in mice treated with the EGFR inhibitor AG1478. Inhibition of EGFR activity greatly diminished MV-induced lung alveolar permeability and neutrophil accumulation in the bronchoalveolar lavage (BAL) fluid, as compared with vehicle-treated controls. Similarly, AG1478 inhibition diminished lung vascular leak (as assessed by Evans blue extravasation), but it did not affect interstitial neutrophil accumulation. Inhibition of the EGFR pathway also blocked expression of genes induced by MV. However, intratracheal instillation of EGF alone failed to induce lung injury. Collectively, our findings suggest that EGFR-activated signaling is necessary but not sufficient to produce acute lung injury in mice.

Genetic and pharmacologic evidence links oxidative stress to ventilator-induced lung injury in mice.

RATIONALE: Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear. OBJECTIVES: We hypothesized that disruption of the gene encoding Nrf2, a transcription factor that regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI) and that antioxidant supplementation attenuates this effect. METHODS: To test our hypothesis and to examine the relevance of oxidative stress in VILI, we assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2(-/-)) mice and wild-type (Nrf2(+/+)) mice after an acute (2-h) injurious model of MV with or without administration of antioxidant. MEASUREMENTS AND MAIN RESULTS: Nrf2(-/-) mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared with Nrf2(+/+) mice. Nrf2 deficiency enhances the levels of several proinflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2(-/-) mice; however, antioxidant supplementation to Nrf2(-/-) mice remarkably attenuated VILI. When subjected to a clinically relevant prolong period of MV, Nrf2(-/-) mice displayed greater levels of VILI than Nrf2(+/+) mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins, and phosphatases in Nrf2(-/-) mice. CONCLUSIONS: Our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress.

Genetic dissection of the Nrf2-dependent redox signaling-regulated transcriptional programs of cell proliferation and cytoprotection.

The beta zipper (bZip) transcription factor, nuclear factor erythroid 2, like 2 (Nrf2), acting via an antioxidant/electrophile response element, regulates the expression of several antioxidant enzymes and maintains cellular redox homeostasis. Nrf2 deficiency diminishes pulmonary expression of several antioxidant enzymes, rendering them highly susceptible to various mouse models of prooxidant-induced lung injury. We recently demonstrated that Nrf2 deficiency impairs primary cultured pulmonary epithelial cell proliferation and greatly enhances sensitivity to prooxidant-induced cell death. Glutathione (GSH) supplementation rescued cells from these defects associated with Nrf2 deficiency. To further delineate the mechanisms by which Nrf2, via redox signaling, regulates cellular protection and proliferation, we compared the global expression profiling of Nrf2-deficient cells with and without GSH supplementation. We found that GSH regulates the expression of various networks of transcriptional programs including 1) several antioxidant enzymes involved in cellular detoxification of reactive oxygen species and recycling of thiol status and 2) several growth factors, growth factor receptors, and integrins that are critical for cell growth and proliferation. We also found that Nrf2 deficiency enhances the expression levels of several genes encoding proinflammatory cytokines; however, GSH supplementation markedly suppressed their expression. Collectively, these findings uncover an important insight into the nature of genes regulated by Nrf2-dependent redox signaling through GSH that are involved in cellular detoxification and proliferation.

Deficiency in Nrf2-GSH signaling impairs type II cell growth and enhances sensitivity to oxidants.

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.