Rationale: Sepsis syndrome is characterized by inappropriate amplified systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Nuclear factor–erythroid 2 p45-related factor 2 (Nrf2) regulates a pleiotropic cytoprotective defense program including antioxidants and protects against several inflammatory disorders by inhibiting oxidative tissue injuries. However, the role of enhanced Nrf2 activity in modulating innate immune responses to microbial infection and pathogenesis of sepsis is unclear.

Objectives: To determine whether Nrf2 in myeloid leukocytes alters inflammatory response and protects against sepsis.

Methods: Mice with deletion of Nrf2 or kelch-like ECH-associated protein (Keap1) in myeloid leukocyte cells and respective floxed controls were subjected to cecal ligation and puncture–induced sepsis and were assessed for survival, organ injury, systemic inflammation, and bacteremia. Using LPS-stimulated peritoneal macrophages, Toll-like receptor (TLR) 4 surface trafficking and downstream signaling events were analyzed.

Measurements and Main Results: Mortality, organ injury, circulating levels of inflammatory mediators, and bacteremia were markedly reduced in LysM-Keap1−/− compared with respective floxed controls (Keap1f/f or Nrf2f/f) and significantly elevated in LysM-Nrf2−/− mice after cecal ligation and puncture. Peritoneal macrophages from septic LysM-Keap1−/− mice showed a greater bacterial phagocytic activity compared with LysM-Nrf2−/− and floxed controls. LPS stimulation resulted in greater reactive oxygen species–induced cell surface transport of TLR4 from trans-Golgi network and subsequent TLR4 downstream signaling (recruitment of MYD88 and TRIF, phosphorylation of IkB and IRF3, and cytokine expression) in macrophages of LysM-Nrf2−/− compared with LysM-Keap1−/− mice and floxed controls.

Conclusions: Our study shows that Nrf2 acts as a critical immunomodulator in leukocytes, controls host inflammatory response to bacterial infection, and protects against sepsis.

Background:

Sulforaphane (SFN), an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), is a promising chemopreventive agent which is undergoing clinical trial for several diseases. Studies have indicated that there is gain of Nrf2 function in lung cancer and other solid tumors because of mutations in the inhibitor Kelch-like ECH-associated protein 1 (Keap1). More recently, several oncogenes have been shown to activate Nrf2 signaling as the main prosurvival pathway mediating ROS detoxification, senescence evasion, and neoplastic transformation. Thus, it is important to determine if there is any risk of enhanced lung tumorigenesis associated with prolonged administration of SFN using mouse models of cancer.

Materials and Methods:

We evaluated the effect of prolonged SFN treatment on oncogenic K-ras (K-rasLSL-G12D)-driven lung tumorigenesis. One week post mutant-K-ras expression, mice were treated with SFN (0.5 mg, 5 d/wk) for 3 months by means of a nebulizer. Fourteen weeks after mutant K-ras expression (K-rasLSL-G12D), mice were sacrificed, and lung sections were screened for neoplastic foci. Expression of Nrf2-dependent genes was measured using real time RT-PCR. We also determined the effect of prolonged SFN treatment on the growth of preclinical xenograft models using human A549 (with mutant K-ras and Keap1 allele) and H1975 [with mutant epidermal growth factor receptor (EGFR) allele] nonsmall cell lung cancer cells.

Results:

Systemic SFN administration did not promote the growth of K-rasLSL-G12D-induced lung tumors and had no significant effect on the growth of A549 and H1975 established tumor xenografts in nude mice. Interestingly, localized delivery of SFN significantly attenuated the growth of A549 tumors in nude mice, suggesting an Nrf2-independent antitumorigenic activity of SFN.

Conclusions:

Our results demonstrate that prolonged SFN treatment does not promote lung tumorigenesis in various mouse models of lung cancer.

Pseudohypoparathyroidism is rare during pregnancy and poses multiple challenges related to its diagnosis and management during pregnancy. We hereby report a case of a young woman who was diagnosed to have type 1a pseudohypoparathyroidism. She was managed by multidisciplinary team and had good maternal and perinatal outcome. Management-related issues are discussed here in detail.

Distinctive histological variants of lung cancer are increasingly recognized to have specific genetic changes that impact tumor biology and response to therapy. In this study, we evaluated true papillary adenocarcinoma of the lung, proposed as a distinct diagnostic category with relatively poor response to therapy, to determine whether these tumors also have specific molecular alterations that would affect sensitivity to chemotherapy. Specifically, we measured protein levels of P53, ERCC1 and RRM1 by immunohistochemistry and evaluated the KEAP1 gene for mutations, correlating mutations of this gene with total and nuclear expression of the NRF2 transcription factor. We found high levels of P53 in 23 of the 55 specimens (41.8%), similar to the rate of P53 gene mutations observed in general for pulmonary adenocarcinoma, and levels of ERCC1 and RRM1 also showed distributions similar to those reported generally for NSCLC. However, KEAP1 alterations were observed at a significantly higher frequency in papillary adenocarcinoma tumors (60%) than what has been reported previously for NSCLC (3% to 19%). These mutations of KEAP1 were associated with increased nuclear accumulation of NRF2 in tumors, as expected for functional alterations. Thus, high rates of KEAP1 mutations and NRF2 overexpression in true papillary adenocarcinoma could be related to poor prognosis and chemotherapy resistance. Furthermore, this distinctive molecular characteristic supports the recognition of true papillary adenocarcinoma as a diagnostic entity.

Mutations in the coactivator CREB-binding protein (CBP) are a major cause of the human skeletal dysplasia Rubinstein-Taybi syndrome (RTS); however, the mechanism by which these mutations affect skeletal mineralization and patterning is unknown. Here, we report the identification of 3-phosphoinositide-dependent kinase 1 (PDK1) as a key regulator of CBP activity and demonstrate that its functions map to both osteoprogenitor cells and mature osteoblasts. In osteoblasts, PDK1 activated the CREB/CBP complex, which in turn controlled runt-related transcription factor 2 (RUNX2) activation and expression of bone morphogenetic protein 2 (BMP2). These pathways also operated in vivo, as evidenced by recapitulation of RTS spectrum phenotypes with osteoblast-specific Pdk1 deletion in mice (Pdk1osx mice) and by the genetic interactions observed in mice heterozygous for both osteoblast-specific Pdk1 deletion and either Runx2 or Creb deletion. Finally, treatment of Pdk1osx and Cbp+/– embryos with BMPs in utero partially reversed their skeletal anomalies at birth. These findings illustrate the in vivo function of the PDK1-AKT-CREB/CBP pathway in bone formation and provide proof of principle for in utero growth factor supplementation as a potential therapy for skeletal dysplasias.

ATP-binding cassette, sub-family G, member 2 (ABCG2) is expressed in both normal and cancer cells, and plays a crucial role in the side population (SP) formation and efflux of xenobiotics and drugs. Nrf2, a redox sensing transcription factor, upon constitutive activation in non-small-cell lung cancer cells up-regulates a wide spectrum of genes involved in redox balance, glutathione metabolism, and drug detoxification that contribute to chemoresistance and tumorigenecity. This study examined the mechanism underlying Nrf2-dependent expression of ABCG2 and its role in multidrug resistance phenotype. In silico analysis of the 5’-promoter flanking region of ABCG2 identified an antioxidant response element at -431 bp to -420 bp. A detailed promoter analysis using luciferase reporter assays demonstrated that antioxidant response element (ARE) at -431 bp to -420 bp is critical for the Nrf2-mediated expression in lung cancer cells. Electrophoresis mobility shift assays (EMSA) and chromatin-immunoprecipitation (ChIP) assays revealed that Nrf2 interacts with ABCG2 ARE element at -431 bp to -420 bp in vitro and in vivo. Disruption of Nrf2 expression in lung cancer and prostate cancer cells, by short hairpin RNA, attenuated the expression of ABCG2 transcript and protein and dramatically reduced the SP fraction in Nrf2-depleted cancer cells. Moreover, depleted levels of ABCG2 in these Nrf2-knockdown cells sensitized them to mitoxantrone and topotecan, two chemotherapy drugs detoxified mainly by ABCG2. As expected, overexpression of Nrf2 cDNA in lung epithelial cells led to an increase in ABCG2 expression and a 2-fold higher SP fraction. Thus, Nrf2-mediated regulation of ABCG2 expression maintains SP fraction and confers chemoresistance.

Background

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.

Methods/Findings

SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.

Conclusion

F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

Background

The upper respiratory tract functions to protect lower respiratory structures from chemical and biological agents in inspired air. Cellular oxidative stress leading to acute and chronic inflammation contributes to the resultant pathology in many of these exposures and is typical of allergic disease, chronic sinusitis, pollutant exposure, and bacterial and viral infections. Little is known about the effective means by which topical treatment of the nose can strengthen its antioxidant and anti-inflammatory defenses. The present study was undertaken to determine if naturally-occurring plant oils with reported antioxidant activity can provide mechanisms through which upper respiratory protection might occur.

Methods

Controlled exposure of the upper respiratory system to ozone and nasal biopsy were carried out in healthy human subjects to assess mitigation of the ozone-induced inflammatory response and to assess gene expression in the nasal mucosa induced by a mixture of five naturally-occurring antioxidant oils - aloe, coconut, orange, peppermint and vitamin E. Cells of the BEAS-2B and NCI-H23 epithelial cell lines were used to investigate the source and potential intracellular mechanisms of action responsible for oil-induced anti-inflammatory activity.

Results

Aerosolized pretreatment with the mixed oil preparation significantly attenuated ozone-induced nasal inflammation. Although most oil components may reduce oxidant stress by undergoing reduction, orange oil was demonstrated to have the ability to induce long-lasting gene expression of several antioxidant enzymes linked to Nrf2, including HO-1, NQO1, GCLm and GCLc, and to mitigate the pro-inflammatory signaling of endotoxin in cell culture systems. Nrf2 activation was demonstrated. Treatment with the aerosolized oil preparation increased baseline levels of nasal mucosal HO-1 expression in 9 of 12 subjects.

Conclusions

These data indicate that selected oil-based antioxidant preparations can effectively reduce inflammation associated with oxidant stress-related challenge to the nasal mucosa. The potential for some oils to activate intracellular antioxidant pathways may provide a powerful mechanism through which effective and persistent cytoprotection against airborne environmental exposures can be provided in the upper respiratory mucosa.

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.

Induction of potent T-cell memory is the goal of vaccinations, but the molecular mechanisms that regulate the formation of memory CD8 T cells are not well understood. Despite the recognition that controls of cellular proliferation and apoptosis govern the number of memory T cells, the cell cycle regulatory mechanisms that control these key cellular processes in CD8 T cells during an immune response are poorly defined. Here, we have identified the cyclin-dependent kinase inhibitor p27Kip1 as a critical regulator of the CD8 T-cell homeostasis at all phases of the T-cell response to an acute viral infection in mice. By acting as a timer for cell cycle exit, p27Kip1 curtailed the programmed expansion of interleukin-2-producing memory precursors and markedly limited the magnitude and quality of CD8 T-cell memory. In the absence of p27Kip1, CD8 T cells showed superior recall responses shortly after vaccination with recombinant Listeria monocytogenes. Additionally, we show that p27Kip1 constrains proliferative renewal of memory CD8 T cells, especially of the effector memory subset. These findings provide critical insights into the cell cycle regulation of CD8 T-cell homeostasis and suggest that modulation of p27Kip1 could bolster vaccine-induced T-cell memory and protective immunity.

Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small-cell lung cancer (NSCLC). In this study, we demonstrate that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth and results in increased sensitivity to chemotherapeutic drug induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.

Loss-of-function mutations in the nuclear factor erythroid-2 related factor-2 (Nrf2) inhibitor, Kelch-like-ECH-associated protein (Keap1), result in increased Nrf2 activity in non–small-cell lung cancer (NSCLC) and confer therapeutic resistance. We detected point mutations in Keap1 gene leading to non-conservative amino acid substitutions in prostate cancer cells. We found novel transcriptional and post-transcriptional mechanisms of Keap1 inactivation such as promoter CpG island hypermethylation and aberrant splicing of Keap1 in DU-145 cells. Very low levels of Keap1 mRNA were detected in DU-145 cells, which significantly increased by treatment with DNA methyltransferase inhibitor 5-aza-cytidine. The loss of Keap1 function led to an enhanced activity of Nrf2 and its downstream electrophile/drug detoxification pathway. Inhibition of Nrf2 expression in DU-145 cells by RNAi attenuated the expression of glutathione, thioredoxin, and the drug efflux pathways involved in counteracting electrophiles, oxidative stress, and detoxification of a broad spectrum of drugs. DU-145 cells expressing Nrf2-shRNA had lower levels of total glutathione and higher levels of intracellular reactive oxygen species. Attenuation of Nrf2 function in DU-145 cells enhanced sensitivity to chemotherapeutic drugs and radiation-induced cell death. In addition, Inhibition of Nrf2 greatly suppressed in vitro and in vivo tumor growth of DU-145 prostate cancer cells. Thus, targeting Nrf2 pathway in prostate cancer cells may provide a novel strategy to enhance chemo- and radio-therapy responsiveness and ameliorate the growth and tumorigenecity leading to improved clinical outcomes.

Abstract

Nuclear factor erythroid-2 related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates the expression of antioxidant enzymes and several anti-apoptotic proteins, which confer cytoprotection against oxidative stress and apoptosis. Constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. In this study, we show that RNAi-mediated lowering of Nrf2 levels in non-small-cell lung cancer (NSCLC) cell lines (A549 and H460) led to a dramatic increase in endogenous reactive oxygen species (ROS) levels. Similarly, γ-irradiation-induced formation of protein carbonyls were significantly higher in Nrf2-depleted lung cancer cells, suggesting increased lethality of ionizing radiation in the absence of Nrf2. Radiation-induced protein oxidation in Nrf2shRNA cells correlated with reduced survival as measured by clonogenic assay. Radiation-induced cell death was abrogated by pretreatment with antioxidants such as N-acetyl-L-cysteine, glutathione, and vitamin-E, highlighting the importance of antioxidants in conferring protection against radiation injury. Using genetically-modified gain and loss of function models of Nrf2, in mouse embryonic fibroblasts, we establish that constitutive activation of Nrf2 protects against ionizing radiation toxicity and confers radioresistance. Thus, targeting Nrf2 activity in radioresistant tumors could be a promising strategy to circumvent radioresistance. Antioxid. Redox Signal. 13, 1627–1637.

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1−/−) or depletion (Nrf2−/−) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response.

Oxidative stress results in protein oxidation and is involved in the pathogenesis of lung diseases such as chronic obstructive pulmonary disorder (COPD). Sulfiredoxin-1 (Srx1) catalyzes reduction of cysteine sulfinic acid to sulfenic acid in oxidized proteins and protects them from inactivation. This study examined the mechanism of transcriptional regulation of Srx1 and its possible protective role during oxidative stress associated with COPD. Nrf2, a transcription factor known to influence susceptibility to pulmonary diseases, upregulates Srx1 expression during oxidative stress caused by cigarette smoke exposure in the lungs of mice. Disruption of Nrf2 signaling by genetic knockout in mice or RNAi in cells downregulated the expression of Srx1. In silico analysis of the 5′-promoter flanking region of Srx1 identified multiple antioxidant response elements that are highly conserved. Reporter and chromatin-immunoprecipation assays demonstrated that ARE1 at −228 is critical for the Nrf2-mediated response. Attenuation of Srx1 expression with RNAi potentiated the toxicity of hydrogen peroxide (H2O2), whereas overexpression of Srx1 protected against H2O2 mediated cell death in vitro. Immunoblot analysis revealed dramatic decreases in Srx1 expression in lungs from patients with COPD relative to non-emphysematous lungs together with a decline in Nrf2 protein. Thus, Srx1, a key Nrf2-regulated gene, contributes to protection against oxidative injury in the lung.

Rationale: Oxidative stress is a key contributor in chronic obstructive pulmonary disease (COPD) pathogenesis caused by cigarette smoking. NRF2, a redox-sensitive transcription factor, dissociates from its inhibitor, KEAP1, to induce antioxidant expression that inhibits oxidative stress.

Objectives: To determine the link between severity of COPD, oxidative stress, and NRF2-dependent antioxidant levels in the peripheral lung tissue of patients with COPD.

Methods: We assessed the expression of NRF2, NRF2-dependent antioxidants, regulators of NRF2 activity, and oxidative damage in non-COPD (smokers and former smokers) and smoker COPD lungs (mild and advanced). Cigarette smoke–exposed human lung epithelial cells (Beas2B) and mice were used to understand the mechanisms.

Measurements and Main Results: When compared with non-COPD lungs, the COPD patient lungs showed (1) marked decline in NRF2-dependent antioxidants and glutathione levels, (2) increased oxidative stress markers, (3) significant decrease in NRF2 protein with no change in NRF2 mRNA levels, and (4) similar KEAP1 but significantly decreased DJ-1 levels (a protein that stabilizes NRF2 protein by impairing KEAP1-dependent proteasomal degradation of NRF2). Exposure of Bea2B cells to cigarette smoke caused oxidative modification and enhanced proteasomal degradation of DJ-1 protein. Disruption of DJ-1 in mouse lungs, mouse embryonic fibroblasts, and Beas2B cells lowered NRF2 protein stability and impaired antioxidant induction in response to cigarette smoke. Interestingly, targeting KEAP1 by siRNA or the small-molecule activator sulforaphane restored induction of NRF2-dependent antioxidants in DJ-1–disrupted cells in response to cigarette smoke.

Conclusions: NRF2-dependent antioxidants and DJ-1 expression was negatively associated with severity of COPD. Therapy directed toward enhancing NRF2-regulated antioxidants may be a novel strategy for attenuating the effects of oxidative stress in the pathogenesis of COPD.

Background:

There is a growing consensus about the validity of human personality traits as important dispositions toward feelings and behaviors (Matthews, Deary, & Whiteman, 2003).

Materials and Methods:

Here we examine the reliability of the Hindi translation of the Eysenck Personality Questionnaire-Revised Short Form (EPQR-S; Eysenck, Eysenck, & Barrett, 1985), which consists of 48 items that assess neuroticism, extraversion, psychoticism, and lying. The questionnaire was first translated into Hindi and then back translated. Subsequently, it was administered to 202 students (78 men and 124 women) from Banaras Hindu University. The internal consistency of the scale was evaluated.

Results:

The findings provide satisfactory psychometric properties of the extraversion, neuroticism and lie scales. The psychoticism scale, however, was found to be less satisfactory.

Conclusion:

It can be proposed that due to satisfactory internal consistency scores, the EPQRS-H is a reliable scale for the measurement of various personality traits.

Disruption of NF-E2–related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke–induced emphysema in mice through the transcriptional induction of numerous antioxidant genes. The lungs of Nrf2−/− mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal) and oxidative DNA damage (7,8-dihydro-8-Oxo-2′deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2, Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells. An ARE sequence located in the 5′ promoter–flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified. Mutation of this ARE core sequence completely abolished the activity of promoter–reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that GPX2 is the major oxidative stress–inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2.

Despite the well-recognized importance of CD4 T-cell help in the induction of antibody production and cytotoxic-T-lymphocyte responses, the regulation of CD4 T-cell responses is not well understood. Using mice deficient for TNF receptor I (TNFR I) and/or TNFR II, we show that TNFR I and TNFR II play redundant roles in down regulating the expansion of CD4 T cells during an acute infection of mice with lymphocytic choriomeningitis virus (LCMV). Adoptive transfer experiments using T-cell-receptor transgenic CD4 T cells and studies with mixed bone marrow chimeras indicated that indirect effects and not direct effects on T cells mediated the suppressive function of TNF on CD4 T-cell expansion during the primary response. Further studies to characterize the indirect effects of TNF suggested a role for TNFRs in LCMV-induced deletion of CD11chi dendritic cells in the spleen, which might be a mechanism to limit the duration of antigenic stimulation and CD4 T-cell expansion. Consequent to enhanced primary expansion, there was a substantial increase in the number of LCMV-specific memory CD4 T cells in the spleens of mice deficient for both TNFR I and TNFR II. In summary, our findings suggest that TNFRs down regulate CD4 T-cell responses during an acute LCMV infection by a non-T-cell autonomous mechanism.

Background

Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.

Methods and Findings

Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.

Conclusions

This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.

Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.

Editors' Summary

Background.

Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.

Why Was This Study Done?

To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.

What Did the Researchers Do and Find?

The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.

What Do These Findings Mean?

These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.

US National Cancer Institute information on lung cancer and on cancer treatment

MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer

Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.

The role of tumor necrosis factor (TNF) in regulating various phases of the antiviral T-cell response is incompletely understood. Additionally, despite strong evidence ascribing a role for TNF in protecting against T-cell-dependent autoimmunity, the underlying mechanisms are still obscure. To address these issues, we have investigated the role of tumor necrosis factor receptors (TNFRs) I (p55R) and II (p75R) in regulating CD8 T-cell responses to lymphocytic choriomeningitis virus (LCMV) with wild-type, p55R-deficient (p55−/−), p75R-deficient (p75−/−), and p55R- and p75R-deficient (DKO) mice. Loss of p55R increased the number of memory CD8 T cells to only one of the two immunodominant epitopes, and p75R deficiency had a minimal impact on the T-cell response to LCMV. Strikingly, deficiency of both p55R and p75R had a more dramatic effect on the LCMV-specific CD8 T-cell response; in the DKO mice, as a sequel to enhanced expansion and a reduction in contraction of CD8 T cells, there was a substantial increase in the number of memory CD8 T cells (specific to the two immunodominant epitopes). While the majority of LCMV-specific memory CD8 T cells in wild-type mice were CD62Lhi CCR7hi (central memory), a major proportion of memory CD8 T cells in DKO mice were CD62Llo CCR7hi. TNFR deficiency did not affect the proliferative renewal of memory CD8 T cells. Taken together, these data suggested that TNFRs p55R and p75R have overlapping roles in downregulating CD8 T-cell responses and establishment of immune homeostasis during an acute viral infection.

Description of the root of the plant, Zizyphus mauritiana Linn. the microscopical characters of the powdered root, its behavior on treatment with different chemical reagents, and the fluorescence character under ultraviolet light after treatment were studied to fix some pharmacognostical parameters. Preliminary phytochemical screening on the various extracts of the root of the plant was also performed. These studies were carried out to identify this plant for further research work.