Knockout of the transcription factor Nrf2: Effects on testosterone production by aging mouse Leydig cells.

Aging in rodents and men is associated with reduced serum levels of testosterone and Leydig cell testosterone productions. To further investigate the mechanism by which Leydig cell testosterone production declines, the effect of knocking out Nrf2, a master regulator of phase 2 antioxidant genes, was examined. In wild-type mice, testosterone production and serum testosterone levels remained unchanged through middle age (8 months), but then were reduced significantly by old age (21-24 months). In contrast, serum testosterone levels and Leydig cell testosterone production were reduced significantly in the Nrf2-/- mice as early as middle age, and were reduced further in the aged mice. Reduced steroidogenesis in the knockout mice was associated with reduced antioxidant capacity, and increased expression of protein nitrotyrosine residues, a marker of ROS. These results support the hypothesis that, over time, increases in oxidative stress contribute to or cause the reduced testosterone production that characterizes Leydig cell aging.

Genetic silencing of Nrf2 enhances X-ROS in dysferlin-deficient muscle.

Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation (X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy-a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression.

NRF2-mediated Notch pathway activation enhances hematopoietic reconstitution following myelosuppressive radiation.

A nuclear disaster may result in exposure to potentially lethal doses of ionizing radiation (IR). Hematopoietic acute radiation syndrome (H-ARS) is characterized by severe myelosuppression, which increases the risk of infection, bleeding, and mortality. Here, we determined that activation of nuclear factor erythroid-2-related factor 2 (NRF2) signaling enhances hematopoietic stem progenitor cell (HSPC) function and mitigates IR-induced myelosuppression and mortality. Augmenting NRF2 signaling in mice, either by genetic deletion of the NRF2 inhibitor Keap1 or by pharmacological NRF2 activation with 2-trifluoromethyl-2'-methoxychalone (TMC), enhanced hematopoietic reconstitution following bone marrow transplantation (BMT). Strikingly, even 24 hours after lethal IR exposure, oral administration of TMC mitigated myelosuppression and mortality in mice. Furthermore, TMC administration to irradiated transgenic Notch reporter mice revealed activation of Notch signaling in HSPCs and enhanced HSPC expansion by increasing Jagged1 expression in BM stromal cells. Administration of a Notch inhibitor ablated the effects of TMC on hematopoietic reconstitution. Taken together, we identified a mechanism by which NRF2-mediated Notch signaling improves HSPC function and myelosuppression following IR exposure. Our data indicate that targeting this pathway may provide a countermeasure against the damaging effects of IR exposure.

Tamoxifen-resistant, ER-positive MAC 51 cell line with a high metastatic potential developed from a spontaneous breast cancer mouse model.

We developed and characterized an estrogen-responsive and ER-positive murine breast cancer cell line (MAC51) from a spontaneous breast cancer animal model. These cells are overexpressed with K8, K18 and K19 proteins in an immunofluoresence assay. Upregulation of ER alpha was observed in the immunofluoresence assay, real-time PCR analysis and western blot assay. A colocalization experiment in MAC 51 showed cytoplasmic colocalization of K18 and K19 proteins with ER α. Real-time analysis of tumor samples from engrafted animals, MAC 51, metastatic liver and metastatic ovary revealed overexpression of K8 and K18 compared to the respective controls. A hormone responsive experiment in immunodeficient mice showed highly significant decreases in estrogen and tumor volume after 14 days ovariectomization. The tumorogenicity assay showed higher (3 × 10 (5)) and lower (3 × 10(4)) concentrations of MAC 51 cells that developed tumors within 2 weeks post-transplantation. Tumor morphology and histology resembled a sarcoma pattern but our spontaneous model appeared in an adenocarcinoma pattern. Metastasis to different organs occurred through hematogenous and lymphatic routes. We assessed the potency of the anticancer effect in MAC 51 cells by treating various anticancer drugs with E2, followed by studying apoptotic gene expression profiles. E2 and E2+ tamoxifen-treated cells showed upregulation of apoptotic genes caspase 1, 3, 9, P53 and Bcl-xl but the tamoxifen- and paclitaxel-treated cells did not upregulate the apoptotic genes. Tamoxifen-resistant, ER-positive and high metastatic potential cell lines from murine origin are very rare. Also, estrogen greatly induced apoptosis in this cell line, hence MAC 51 has a greater application potential to evaluate low doses of estrogen with other targeted therapeutic drugs to treat breast cancer.

Prolonged sulforaphane treatment does not enhance tumorigenesis in oncogenic K-ras and xenograft mouse models of lung cancer.

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-ras(LSL-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-ras(LSL-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-ras(LSL-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.

Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients.

Chronic obstructive pulmonary disease (COPD), which is caused primarily by cigarette smoking, is a major health problem worldwide. The progressive decline in lung function that occurs in COPD is a result of persistent inflammation of the airways and destruction of the lung parenchyma. Despite the key role of inflammation in the pathogenesis of COPD, treatment with corticosteroids - normally highly effective antiinflammatory drugs - has little therapeutic benefit. This corticosteroid resistance is largely caused by inactivation of histone deacetylase 2 (HDAC2), which is critical for the transrepressive activity of the glucocorticoid receptor (GR) that mediates the antiinflammatory effect of corticosteroids. Here, we show that in alveolar macrophages from patients with COPD, S-nitrosylation of HDAC2 is increased and that this abolishes its GR-transrepression activity and promotes corticosteroid insensitivity. Cys-262 and Cys-274 of HDAC2 were found to be the targets of S-nitrosylation, and exogenous glutathione treatment of macrophages from individuals with COPD restored HDAC2 activity. Treatment with sulforaphane, a small-molecule activator of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), was also able to denitrosylate HDAC2, restoring dexamethasone sensitivity in alveolar macrophages from patients with COPD. These effects of sulforaphane were glutathione dependent. We conclude that NRF2 is a novel drug target for reversing corticosteroid resistance in COPD and other corticosteroid-resistant inflammatory diseases.

Enhancing Nrf2 pathway by disruption of Keap1 in myeloid leukocytes protects against sepsis.

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 (Keap1(f/f) or Nrf2(f/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.

Nrf2 has a protective role against neuronal and capillary degeneration in retinal ischemia-reperfusion injury.

Retinal ischemia-reperfusion (I/R) involves an extensive increase in reactive oxygen species as well as proinflammatory changes that result in significant histopathologic damage, including neuronal and vascular degeneration. Nrf2 has a well-known cytoprotective role in many tissues, but its protective function in the retina is unclear. We investigated the possible role of Nrf2 as a protective mechanism in retinal ischemia-reperfusion injury using Nrf2(-/-) mice. I/R resulted in an increase in retinal levels of superoxide and proinflammatory mediators, as well as leukocyte infiltration of the retina and vitreous, in Nrf2(+/+) mice. These effects were greatly accentuated in Nrf2(-/-) mice. With regard to histopathologic damage, Nrf2(-/-) mice exhibited loss of cells in the ganglion cell layer and markedly accentuated retinal capillary degeneration, as compared to wild-type. Treatment with the Nrf2 activator CDDO-Me increased antioxidant gene expression and normalized I/R-induced superoxide in the retina in wild-type but not Nrf2(-/-) mice. CDDO-Me treatment abrogated retinal capillary degeneration induced by I/R in wild-type but not Nrf2(-/-) mice. These studies indicate that Nrf2 is an important cytoprotective mechanism in the retina in response to ischemia-reperfusion injury and suggest that pharmacologic induction of Nrf2 could be a new therapeutic strategy for retinal ischemia-reperfusion and other retinal diseases.

NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice.

Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared with wild-type (Nrf2(+/+)) macrophages, we observed greater protein kinase C-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2(-/-)) macrophages that was modulated by glutathione levels. To address the NADPH oxidase-mediated hyperinflammatory response and sepsis-induced lung injury and mortality in Nrf2(-/-) mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91(phox) (Nrf2(-/-)//gp91(phox-/-)). Compared with Nrf2(+/+) macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MyD88 and Toll/IL-1R domain-containing adaptor in Nrf2(-/-) macrophages that was diminished by ablation of gp91(phox). Similarly, phosphorylation of IkappaB and IFN regulatory factor 3 as well as cytokine expression was markedly higher in Nrf2(-/-) macrophages; whereas, it was similar in Nrf2(+/+) and Nrf2(-/-)//gp91(phox-/-). In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2(-/-) mice that was significantly reduced by ablation of gp91(phox). Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2(-/-) mice; whereas, Nrf2(-/-)//gp91(phox-/-) showed prolonged survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.

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.

Deletion of Keap1 in the lung attenuates acute cigarette smoke-induced oxidative stress and inflammation.

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.

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.