Heterocyclic chalcone activators of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with improved in vivo efficacy.

Nrf2 activators represent a good drug target for designing agents to treat diseases associated with oxidative stress. Building upon previous work, we designed and prepared a series of heterocyclic chalcone-based Nrf2 activators with reduced lipophilicity and, in some cases, greater in vitro potency compared to the respective carbocyclic scaffold. These changes resulted in enhanced oral bioavailability and a superior pharmacodynamic effect in vivo.

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.

Hyperoxia stimulates an Nrf2-ARE transcriptional response via ROS-EGFR-PI3K-Akt/ERK MAP kinase signaling in pulmonary epithelial cells.

Nuclear factor erythroid 2-related factor (Nrf2) confers protection against cell death induced by hyperoxia and other proapoptotic stimuli. Because phosphoinositide-3-kinase (PI3K)/Akt signaling promotes cell survival, the significance of this pathway in mediating reactive oxygen species (ROS)-dependent hyperoxia-induced Nrf2 activation was investigated in the murine pulmonary epithelial cell line, C10. Inhibition of the PI3K pathway markedly attenuated hyperoxia-induced Nrf2 translocation and ARE (antioxidant response element)-mediated transcription. Consistent with this, hyperoxia markedly stimulated the activation of PI3K pathway, while an NADPH oxidase inhibitor and an antioxidant prevented such activation. The inhibition of Akt activity using a pharmacological inhibitor markedly attenuated Nrf2 translocation and ARE-driven expression. Moreover, overexpression of a dominant-negative Akt mutant attenuated the transcription, whereas a constitutively active mutant stimulated it. These results suggest that PI3K/Akt signaling regulates Nrf2 activation by hyperoxia. Inhibition of the PI3K pathway prevented hyperoxia-stimulated Akt and ERK1/2 kinase activation, which is critical for Nrf2-mediated transcription. Likewise, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, AG1478, blocked hyperoxia-stimulated Akt and ERK1/2 phosphorylation, Nrf2 nuclear accumulation, and ARE-driven transcription. Consistent with this result, an NADPH oxidase inhibitor blocked hyperoxia- stimulated EGFR phosphorylation, which was correlated with the attenuation of Akt and ERK activation. Collectively, our data suggest that EGFR-PI3K signaling through Akt and ERK kinases regulates ROS-dependent, hyperoxia-induced Nrf2 activation in pulmonary epithelial cells.

African ancestry is associated with asthma risk in African Americans.

BACKGROUND: Asthma is a common complex condition with clear racial and ethnic differences in both prevalence and severity. Asthma consultation rates, mortality, and severe symptoms are greatly increased in African descent populations of developed countries. African ancestry has been associated with asthma, total serum IgE and lower pulmonary function in African-admixed populations. To replicate previous findings, here we aimed to examine whether African ancestry was associated with asthma susceptibility in African Americans. In addition, we examined for the first time whether African ancestry was associated with asthma exacerbations. METHODOLOGY/PRINCIPAL FINDINGS: After filtering for self-reported ancestry and genotype data quality, samples from 1,117 self-reported African-American individuals from New York and Baltimore (394 cases, 481 controls), and Chicago (321 cases followed for asthma exacerbations) were analyzed. Genetic ancestry was estimated based on ancestry informative markers (AIMs) selected for being highly divergent among European and West African populations (95 AIMs for New York and Baltimore, and 66 independent AIMs for Chicago). Among case-control samples, the mean African ancestry was significantly higher in asthmatics than in non-asthmatics (82.0±14.0% vs. 77.8±18.1%, mean difference 4.2% [95% confidence interval (CI):2.0-6.4], p<0.0001). This association remained significant after adjusting for potential confounders (odds ratio: 4.55, 95% CI: 1.69-12.29, p = 0.003). African ancestry failed to show an association with asthma exacerbations (p = 0.965) using a model based on longitudinal data of the number of exacerbations followed over 1.5 years. CONCLUSIONS/SIGNIFICANCE: These data replicate previous findings indicating that African ancestry constitutes a risk factor for asthma and suggest that elevated asthma rates in African Americans can be partially attributed to African genetic ancestry.

Association between lifestyle factors and CpG island methylation in a cancer-free population.

BACKGROUND: Many risk factors have been associated with cancer, such as age, family history, race, smoking, high-fat diet, and poor nutrition. It is important to reveal the molecular changes related to risk factors that could facilitate early detection, prevention, and overall control of cancer. METHODS: We selected six cancer-specific methylated genes that have previously been reported in primary tumors and have also been detected in different bodily fluids of cancer patients. Here, we used quantitative fluorogenic real-time methylation-specific PCR in plasma DNA samples for the detection of methylation changes from an asymptomatic population who do not have any known cancer. RESULTS: The promoter methylation frequencies of the studied genes were as follows: APC (7%), CCND2 (22%), GSTP1 (2%), MGMT (9%), RARbeta2 (29%), and P16 (3%). Promoter methylation of at least one of the genes analyzed was observed in approximately 46% (72 of 157) of the samples by binary dichotomization. Promoter hypermethylation of at least two genes was detected in 17% (26 of 157) of the samples. RARbeta2 methylation was observed in 45% of subjects who had a high-fat diet in contrast with those who had a low-fat diet (23%; P = 0.007). DISCUSSION: Our findings may help to elucidate early methylation changes that may lead to cancer development. These methylation changes could be due to exposure to risk factors and may be useful for cancer prevention measures such as changes in lifestyle. Longitudinal follow-up of a high-risk population is needed to understand the association of methylation of candidate genes in cancer development.

EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced NRF2-ARE activation.

Cyclic stretch (CS) associated with mechanical ventilation (MV) can cause excessive alveolar and endothelial distention, resulting in lung injury and inflammation. Antioxidant enzymes (AOEs) play a major role in suppressing these effects. The transcription factor Nrf2, via the antioxidant response element (ARE), alleviates pulmonary toxicant- and oxidant-induced oxidative stress by up-regulating the expression of several AOEs. Although gene expression profiling has revealed the induction of AOEs in the lungs of rodents exposed to MV, the mechanisms by which mechanical forces, such as CS, regulate the activation of Nrf2-dependent ARE-transcriptional responses are poorly understood. To mimic mechanical stress associated with MV, we have cultured pulmonary alveolar epithelial and endothelial cells on collagen I-coated BioFlex plates and subjected them to CS. CS exposure stimulated ARE-driven transcriptional responses and subsequent AOE expression. Ectopic expression of a dominant-negative Nrf2 suppressed the CS-stimulated ARE-driven responses. Our findings suggest that actin remodeling is necessary but not sufficient for high-level CS-induced ARE activation in both epithelial and endothelial cells. We also found that inhibition of EGFR activity by a pharmacologic agent ablated the CS-induced ARE transcriptional response in both cell types. Additional studies revealed that amphiregulin, an EGFR ligand, regulates this process. We further demonstrated that the PI3K-Akt pathway acts as the downstream effector of EGFR and regulates CS-induced ARE-activation in an oxidative stress-dependent manner. Collectively, these novel findings suggest that EGFR-activated signaling and actin remodeling act in concert to regulate the CS-induced Nrf2-ARE transcriptional response and subsequent AOE expression.

Multiple cis-elements mediate the transcriptional activation of human fra-1 by 12-O-tetradecanoylphorbol-13-acetate in bronchial epithelial cells.

Recent studies indicate a potential role for Fra-1, a heterodimeric partner of activator protein 1 (AP1), in toxicant-induced epithelial injury, repair, and cellular transformation. Here, we have investigated the transcriptional regulation of fra-1 by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human bronchial epithelial (HBE) cells, which are the direct targets of inhaled toxins/carcinogens. In contrast to a transient induction by H2O2, TPA persistently activated fra-1 transcription, principally at the transcriptional level. A deletion analysis of the fra-1 promoter revealed that several cis-elements located between -105/+32 and -283/-105 bp mediate minimal and basal promoter activities, respectively. A region between -379 and -283 bp, which harbors a putative TPA response element, a GC box, and an Ets-like binding site, was required for high level TPA-inducible expression. Mutations in any of these cis-elements markedly reduced both basal and TPA-inducible expression. Thus, cooperative interactions between factors binding to multiple cis-elements of the -379/-283 promoter region appear to regulate TPA-induced fra-1 transcription in HBE cells. Consistent with this finding, electrophoretic mobility shift assays indicated the formation of multiple complexes consisting of the AP1-, Sp-, and ETS-specific family of transcription factors with the -379/-283 fragment. Members of the AP1 family distinctly regulated the fra-1 promoter. In particular, coexpression of c-Jun, Jun-D, and Fra-2 up-regulated fra-1 transcription. Chromatin immunoprecipitation assays revealed an enhanced recruitment of c-Jun, Jun-D, and Fra-2 to the endogenous fra-1 promoter upon TPA stimulation. These results underscore the regulatory role of c-Jun, Jun-D, and Fra-2 in TPA-inducible fra-1 expression in HBE cells in vivo.

NADPH oxidase and ERK signaling regulates hyperoxia-induced Nrf2-ARE transcriptional response in pulmonary epithelial cells.

Oxidative stress plays a major role in hyperoxia-induced acute lung injury. We have shown previously that mice lacking the Nrf2 are more susceptible to hyperoxia than are wild-type mice. Nrf2 activates antioxidant response element (ARE)-mediated gene expression involved in cellular protection against toxic insults. The present study was designed to investigate the mechanisms that control the activation of Nrf2 by hyperoxia using a non-malignant murine alveolar epithelial cell line, C10. No significant alteration in the levels of Nrf2 mRNA and protein was found following exposure to hyperoxia. In contrast, hyperoxia caused the translocation of Nrf2 from the cytoplasm to the nucleus within 30-60 min of exposure. Consistent with these observations, gel shift and reporter analyses demonstrated a correlation between the hyperoxia-enhanced ARE DNA-binding activity of Nrf2 and an up-regulation of ARE-driven transcription. Inhibition of NADPH oxidase with diphenyleneiodonium (DPI) blocked both Nrf2 translocation and ARE-mediated transcription. Inhibition of the MEK/ERK pathway caused a similar effect. Consistent with this finding, hyperoxia stimulated ERK-1 and ERK-2 phosphorylation, whereas DPI or N-acetyl-l-cysteine blocked such activation. Hyperoxia stimulated the phosphorylation of endogenous Nrf2, but not in the presence of U0126, suggesting a critical role for ERK signaling in the activation of Nrf2. Consistent with this notion, hyperoxia did not stimulate the phosphorylation of Nrf2 in fibroblasts lacking the ERK-1. Collectively, our findings suggest that hyperoxia-induced, ARE-driven, Nrf2-dependent transcription is controlled by NADPH oxidase and ERK-1 signaling.