Shyam Biswal

Multiple-laboratory comparison of microarray platforms

Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC–Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.

Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis

Host genetic factors that regulate innate immunity determine susceptibility to sepsis. Disruption of nuclear factor-erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor that regulates redox balance and stress response, dramatically increased the mortality of mice in response to endotoxin- and cecal ligation and puncture-induced septic shock. LPS as well as TNF-alpha stimulus resulted in greater lung inflammation in Nrf2-deficient mice. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as 30 minutes in lungs of Nrf2-deficient mice, indicating severe immune dysregulation. The expression profile indicated that Nrf2 has a global influence on both MyD88-dependent and -independent signaling. Nrf2-deficient mouse embryonic fibroblasts showed greater activation of NF-kappaB and interferon regulatory factor 3 in response to LPS and polyinosinic-polycytidylic acid [poly(I:C)] stimulus, corroborating the effect of Nrf2 on MyD88-dependent and -independent signaling. Nrf2s regulation of cellular glutathione and other antioxidants is critical for optimal NF-kappaB activation in response to LPS and TNF-alpha. Our study reveals Nrf2 as a novel modifier gene of sepsis that determines survival by mounting an appropriate innate immune response.

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

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.

Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis

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.

Decline in NRF2-regulated Antioxidants in Chronic Obstructive Pulmonary Disease Lungs Due to Loss of Its Positive Regulator, DJ-1

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.

Targeting Nrf2 with the triterpenoid CDDO-imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice

Chronic obstructive pulmonary disease (COPD), which comprises emphysema and chronic bronchitis resulting from prolonged exposure to cigarette smoke (CS), is a major public health burden with no effective treatment. Emphysema is also associated with pulmonary hypertension, which can progress to right ventricular failure, an important cause of morbidity and mortality among patients with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a redox-sensitive transcription factor that up-regulates a battery of antioxidative genes and cytoprotective enzymes that constitute the defense against oxidative stress. Recently, it has been shown that patients with advanced COPD have a decline in expression of the Nrf2 pathway in lungs, suggesting that loss of this antioxidative protective response is a key factor in the pathophysiological progression of emphysema. Furthermore, genetic disruption of Nrf2 in mice causes early-onset and severe emphysema. The present study evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with a small molecule would attenuate CS-induced oxidative stress and emphysema. Nrf2+/+ and Nrf2−/− mice were fed a diet containing the potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6 months. CDDO-Im significantly reduced lung oxidative stress, alveolar cell apoptosis, alveolar destruction, and pulmonary hypertension in Nrf2+/+ mice caused by chronic exposure to CS. This protection from CS-induced emphysema depended on Nrf2, as Nrf2−/− mice failed to show significant reduction in alveolar cell apoptosis and alveolar destruction after treatment with CDDO-Im. These results suggest that targeting the Nrf2 pathway during the etiopathogenesis of emphysema may represent an important approach for prophylaxis against COPD.

RNAi-Mediated Silencing of Nuclear Factor Erythroid-2–Related Factor 2 Gene Expression in Non–Small Cell Lung Cancer Inhibits Tumor Growth and Increases Efficacy of Chemotherapy

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. In this study, we show that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation 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 Kelch-Like ECH-Associated Protein 1 Function in Prostate Cancer Cells Causes Chemoresistance and Radioresistance and Promotes Tumor Growth

Loss-of-function mutations in the nuclear factor erythroid-2–related factor 2 (Nrf2) inhibitor Kelch-like ECH-associated protein 1 (Keap1) result in increased Nrf2 activity in non–small cell lung cancer and confer therapeutic resistance. We detected point mutations in Keap1 gene, leading to nonconservative amino acid substitutions in prostate cancer cells. We found novel transcriptional and posttranscriptional 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-deoxycytidine. 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 RNA interference 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 constitutively expressing Nrf2 short hairpin RNA 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 the Nrf2 pathway in prostate cancer cells may provide a novel strategy to enhance chemotherapy and radiotherapy responsiveness and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes. Mol Cancer Ther; 9(2); 336–46

Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2(-/-)) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2(+/+)). Nrf2(-/-) MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2(+/+) MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2(-/-) MEFs compared to Nrf2(+/+) MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, -44; ARE2, -813; ARE3, -1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.

Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by Tetrahydrobiopterin: Efficacy of Recoupling Nitric Oxide Synthase as a Therapeutic Strategy

Background— Sustained pressure overload induces pathological cardiac hypertrophy and dysfunction. Oxidative stress linked to nitric oxide synthase (NOS) uncoupling may play an important role. We tested whether tetrahydrobiopterin (BH4) can recouple NOS and reverse preestablished advanced hypertrophy, fibrosis, and dysfunction. Methods and Results— C57/Bl6 mice underwent transverse aortic constriction for 4 weeks, increasing cardiac mass (190%) and diastolic dimension (144%), lowering ejection fraction (−46%), and triggering NOS uncoupling and oxidative stress. Oral BH4 was then administered for 5 more weeks of pressure overload. Without reducing loading, BH4 reversed hypertrophy and fibrosis, recoupled endothelial NOS, lowered oxidant stress, and improved chamber and myocyte function, whereas untreated hearts worsened. If BH4 was started at the onset of pressure overload, it did not suppress hypertrophy over the first week when NOS activity remained preserved even in untreated transverse aortic constriction hearts. However, BH4 stopped subsequent remodeling when NOS activity was otherwise declining. A broad antioxidant, Tempol, also reduced oxidant stress yet did not recouple NOS or reverse worsened hypertrophy/fibrosis from sustained transverse aortic constriction. Microarray analysis revealed very different gene expression profiles for both treatments. BH4 did not enhance net protein kinase G activity. Finally, transgenic mice with enhanced BH4 synthesis confined to endothelial cells were unprotected against pressure overload, indicating that exogenous BH4 targeted myocytes and fibroblasts. Conclusions— NOS recoupling by exogenous BH4 ameliorates preexisting advanced cardiac hypertrophy/fibrosis and is more effective than a less targeted antioxidant approach (Tempol). These data highlight the importance of myocyte NOS uncoupling in hypertrophic heart disease and support BH4 as a potential new approach to treat this disorder.