RESUMO
Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances ß-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or ß-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the ß-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and ß-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or ß-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.
Assuntos
Quinase 3 da Glicogênio Sintase , Células Secretoras de Insulina , Animais , Camundongos , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Culina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Estabilidade Proteica , Transcrição GênicaRESUMO
It is widely accepted that activating the transcription factor NRF2 will blast the physiological anti-inflammatory mechanisms, which will help combat pathologic inflammation. Much effort is being put in inhibiting the main NRF2 repressor, KEAP1, with either electrophilic small molecules or disrupters of the KEAP1/NRF2 interaction. However, targeting ß-TrCP, the non-canonical repressor of NRF2, has not been considered yet. After in silico screening of â¼1 million compounds, we now describe a novel small molecule, PHAR, that selectively inhibits the interaction between ß-TrCP and the phosphodegron in transcription factor NRF2. PHAR upregulates NRF2-target genes such as Hmox1, Nqo1, Gclc, Gclm and Aox1, in a KEAP1-independent, but ß-TrCP dependent manner, breaks the ß-TrCP/NRF2 interaction in the cell nucleus, and inhibits the ß-TrCP-mediated in vitro ubiquitination of NRF2. PHAR attenuates hydrogen peroxide induced oxidative stress and, in lipopolysaccharide-treated macrophages, it downregulates the expression of inflammatory genes Il1b, Il6, Cox2, Nos2. In mice, PHAR selectively targets the liver and greatly attenuates LPS-induced liver inflammation as indicated by a reduction in the gene expression of the inflammatory cytokines Il1b, TNf, and Il6, and in F4/80-stained liver resident macrophages. Thus, PHAR offers a still unexplored alternative to current NRF2 activators by acting as a ß-TrCP/NRF2 interaction inhibitor that may have a therapeutic value against undesirable inflammation.
Assuntos
Ubiquitina-Proteína Ligases , Proteínas Contendo Repetições de beta-Transducina , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Interleucina-6/metabolismo , Fígado/metabolismo , InflamaçãoRESUMO
The management of locally advanced muscle invasive bladder cancer (MIBC) often necessitates neo-adjuvant chemotherapy (NAC) to eliminate any micro-metastatic disease prior to definitive radical cystectomy (RC) and pelvic lymph node dissection (PLND). The most common imaging techniques traditionally used during this process are computerised tomography (CT) and magnetic resonance imaging (MRI), both of which lack a high sensitivity for nodal staging. In this paper, we attempt to review the evolving indications of F-fluoro-2-deoxy-D-glucose positron emission tomography/computerised tomography (FDG-PET/CT) imaging, in the pre-clinical and post-treatment staging of bladder cancer, with a focus on its ability to evaluate response to NAC. We concluded that use of FDG-PET/CT allows for improved nodal staging and metastatic disease detection, compared to traditional imaging modalities. This enabled earlier detection of tumour response to NAC and/or residual disease, impacting factors such as duration of chemotherapy, with its associated adverse effects, and timing of surgical intervention. However, further studies are required to reliably assess its impact on both overall and disease-free survival.
Assuntos
Fluordesoxiglucose F18 , Neoplasias da Bexiga Urinária , Fluordesoxiglucose F18/uso terapêutico , Humanos , Linfonodos/patologia , Metástase Linfática/patologia , Estadiamento de Neoplasias , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/uso terapêutico , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/terapiaRESUMO
Various techniques exist for treating recurrent carpal and ulnar tunnel syndrome, but AxoGuard nerve wrap has shown promising results for treatment of compression neuropathies when used in conjunction with neurolysis and tenosynovectomy. Prior results demonstrate no safety concerns, and there have not been any reported cases of infection, persistent inflammation, or recurrent perineural fibrosis. A 41-year-old, right-hand-dominant woman experienced repeated bouts of carpal and ulnar tunnel syndromes, which were treated with a small intestine submucosa matrix wrap around the median and ulnar nerves in the wrist. Here, we report a case of necrotic granulomatous inflammation 2.5 months after AxoGuard xenograft nerve wrap was placed around the median and ulnar nerves. As a salvage, NuShield placental allograft was wrapped around the median nerve, which has shown promising results at several months follow-up. Placental allograft nerve wraps represent a useful tool in compression neuropathy resistant to autografts, xenografts, and revision decompression operations.
RESUMO
The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy.
Assuntos
Astrócitos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Antioxidantes , Astrócitos/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Estresse OxidativoRESUMO
NF-E2 p45-related factor 2 (NRF2, encoded in the human by NFE2L2) mediates short-term adaptation to thiol-reactive stressors. In normal cells, activation of NRF2 by a thiol-reactive stressor helps prevent, for a limited period of time, the initiation of cancer by chemical carcinogens through induction of genes encoding drug-metabolising enzymes. However, in many tumour types, NRF2 is permanently upregulated. In such cases, its overexpressed target genes support the promotion and progression of cancer by suppressing oxidative stress, because they constitutively increase the capacity to scavenge reactive oxygen species (ROS), and they support cell proliferation by increasing ribonucleotide synthesis, serine biosynthesis and autophagy. Herein, we describe cancer chemoprevention and the discovery of the essential role played by NRF2 in orchestrating protection against chemical carcinogenesis. We similarly describe the discoveries of somatic mutations in NFE2L2 and the gene encoding the principal NRF2 repressor, Kelch-like ECH-associated protein 1 (KEAP1) along with that encoding a component of the E3 ubiquitin-ligase complex Cullin 3 (CUL3), which result in permanent activation of NRF2, and the recognition that such mutations occur frequently in many types of cancer. Notably, mutations in NFE2L2, KEAP1 and CUL3 that cause persistent upregulation of NRF2 often co-exist with mutations that activate KRAS and the PI3K-PKB/Akt pathway, suggesting NRF2 supports growth of tumours in which KRAS or PKB/Akt are hyperactive. Besides somatic mutations, NRF2 activation in human tumours can occur by other means, such as alternative splicing that results in a NRF2 protein which lacks the KEAP1-binding domain or overexpression of other KEAP1-binding partners that compete with NRF2. Lastly, as NRF2 upregulation is associated with resistance to cancer chemotherapy and radiotherapy, we describe strategies that might be employed to suppress growth and overcome drug resistance in tumours with overactive NRF2.
RESUMO
Contingent upon concentration, reactive oxygen species (ROS) influence cancer evolution in apparently contradictory ways, either initiating/stimulating tumorigenesis and supporting transformation/proliferation of cancer cells or causing cell death. To accommodate high ROS levels, tumor cells modify sulfur-based metabolism, NADPH generation, and the activity of antioxidant transcription factors. During initiation, genetic changes enable cell survival under high ROS levels by activating antioxidant transcription factors or increasing NADPH via the pentose phosphate pathway (PPP). During progression and metastasis, tumor cells adapt to oxidative stress by increasing NADPH in various ways, including activation of AMPK, the PPP, and reductive glutamine and folate metabolism.
Assuntos
Antioxidantes/metabolismo , Glucose/metabolismo , NADP/metabolismo , Neoplasias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/genética , Metabolismo Energético/genética , Humanos , Neoplasias/genéticaRESUMO
3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen present in diesel exhaust. It requires metabolic activation via nitroreduction in order to form DNA adducts and promote mutagenesis. We have determined that human aldo-keto reductases (AKR1C1-1C3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines and collectively represent 50% of the nitroreductase activity. The genes encoding these enzymes are induced by the transcription factor NF-E2 p45-related factor 2 (NRF2), which raises the possibility that NRF2 activation exacerbates 3-NBA toxification. Since A549 cells possess constitutively active NRF2, we examined the effect of heterozygous (NRF2-Het) and homozygous NRF2 knockout (NRF2-KO) by CRISPR-Cas9 gene editing on the activation of 3-NBA. To evaluate whether NRF2-mediated gene induction increases 3-NBA activation, we examined the effects of NRF2 activators in immortalized human bronchial epithelial cells (HBEC3-KT). Changes in AKR1C1-1C3 and NQO1 expression by NRF2 knockout or use of NRF2 activators were confirmed by qPCR, immunoblots, and enzyme activity assays. We observed decreases in 3-NBA activation in the A549 NRF2 KO cell lines (53% reduction in A549 NRF2-Het cells and 82% reduction in A549 NRF2-KO cells) and 40-60% increases in 3-NBA bioactivation due to NRF2 activators in HBEC3-KT cells. Together, our data suggest that activation of the transcription factor NRF2 exacerbates carcinogen metabolism following exposure to diesel exhaust which may lead to an increase in 3-NBA-derived DNA adducts.
Assuntos
Poluentes Atmosféricos/toxicidade , Benzo(a)Antracenos/toxicidade , Regulação da Expressão Gênica/fisiologia , Mutagênicos/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , 20-Hidroxiesteroide Desidrogenases/genética , Células A549 , Ativação Metabólica , Poluentes Atmosféricos/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Benzo(a)Antracenos/metabolismo , Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Hidroxiesteroide Desidrogenases/genética , Imidazóis/farmacologia , Isotiocianatos/farmacologia , Mutagênicos/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SulfóxidosRESUMO
The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), are critical in the maintenance of redox, metabolic and protein homeostasis, as well as the regulation of inflammation. Thus, NRF2 activation provides cytoprotection against numerous pathologies including chronic diseases of the lung and liver; autoimmune, neurodegenerative and metabolic disorders; and cancer initiation. One NRF2 activator has received clinical approval and several electrophilic modifiers of the cysteine-based sensor KEAP1 and inhibitors of its interaction with NRF2 are now in clinical development. However, challenges regarding target specificity, pharmacodynamic properties, efficacy and safety remain.
Assuntos
Tratamento Farmacológico , Proteína 1 Associada a ECH Semelhante a Kelch/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Ativação Metabólica , Animais , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.
RESUMO
In this issue of Cancer Cell, Ge et al. show that overexpression of the oncoprotein iASPP in cancer cells provokes NRF2-mediated induction of cytoprotective genes, because it logjams the ubiquitin ligase substrate adaptor function of KEAP1 by virtue of the fact that it possesses a novel DLT-containing KEAP1-interaction motif.
Assuntos
Fator 2 Relacionado a NF-E2 , Neoplasias , Proteínas Adaptadoras de Transdução de Sinal , Resistência a Medicamentos , Humanos , Proteína 1 Associada a ECH Semelhante a KelchRESUMO
In this issue of Molecular Cell, Chen et al. (2017) demonstrate that the tumor suppressor protein ARF sensitizes cancer cells to programmed death through a surprising mechanism: ARF physically interacts with and antagonizes activation by acetylation of the master redox regulator NRF2, providing an unusual mode of posttranslational NRF2 regulation.
Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2/genética , Epigenômica , Humanos , Oxirredução , Processamento de Proteína Pós-TraducionalRESUMO
We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.
Assuntos
Carcinogênese/genética , Colangiocarcinoma/genética , Glutationa Transferase/genética , Estresse Oxidativo/efeitos dos fármacos , Aflatoxina B1/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/fisiopatologia , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP3A/genética , Adutos de DNA/efeitos dos fármacos , F2-Isoprostanos/administração & dosagem , Feminino , Glutationa Transferase/biossíntese , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Caracteres SexuaisRESUMO
This corrects the article DOI: 10.1038/bjc.2016.363.
RESUMO
The transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, with gene called NFE2L2) is a master regulator of the antioxidant response. In the last decade, interest has intensified in this research area as its importance in several physiological and pathological processes has become widely recognized; these include redox signalling and redox homoeostasis, drug metabolism and disposition, intermediary metabolism, cellular adaptation to stress, chemoprevention and chemoresistance, toxicity, inflammation, neurodegeneration, lipogenesis and aging. Regulation of Nrf2 is complex and although much attention has focussed on its repression by Kelch-like ECH-associated protein-1 (Keap1), recently it has become increasingly apparent that it is also controlled by cross-talk with other signalling pathways including the glycogen synthase kinase-3 (GSK-3)-ß-transducin repeat-containing protein (ß-TrCP) axis, ERAD (endoplasmic reticulum-associated degradation)-associated E3 ubiquitin-protein ligase (Hrd1, also called synoviolin), nuclear factor-kappa B (NF-κB), Notch and AMP kinase. Due to its beneficial role in several diseases, Nrf2 has become a major therapeutic target, with novel natural, synthetic and targeted small molecules currently under investigation to modulate the pathway and in clinical trials.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Congressos como Assunto , Homeostase , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Oxirredução , Transdução de SinaisRESUMO
Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and stress-inducible expression of a battery of genes encoding key components of the glutathione-based and thioredoxin-based antioxidant systems, as well as aldo-keto reductase, glutathione S-transferase, and NAD(P)H: quinone oxidoreductase-1 drug-metabolizing isoenzymes along with multidrug-resistance-associated efflux pumps. It therefore plays a pivotal role in both intrinsic resistance and cellular adaptation to reactive oxygen species (ROS) and xenobiotics. Activation of Nrf2 can, however, serve as a double-edged sword because some of the genes it induces may contribute to chemical carcinogenesis by promoting futile redox cycling of polycyclic aromatic hydrocarbon metabolites or confer resistance to chemotherapeutic drugs by increasing the expression of efflux pumps, suggesting its cytoprotective effects will vary in a context-specific fashion. In addition to cytoprotection, Nrf2 also controls genes involved in intermediary metabolism, positively regulating those involved in NADPH generation, purine biosynthesis, and the ß-oxidation of fatty acids, while suppressing those involved in lipogenesis and gluconeogenesis. Nrf2 is subject to regulation at multiple levels. Its ability to orchestrate adaptation to oxidants and electrophiles is due principally to stress-stimulated modification of thiols within one of its repressors, the Kelch-like ECH-associated protein 1 (Keap1), which is present in the cullin-3 RING ubiquitin ligase (CRL) complex CRLKeap1. Thus modification of Cys residues in Keap1 blocks CRLKeap1 activity, allowing newly translated Nrf2 to accumulate rapidly and induce its target genes. The ability of Keap1 to repress Nrf2 can be attenuated by p62/sequestosome-1 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent manner, thereby allowing refeeding after fasting to increase Nrf2-target gene expression. In parallel with repression by Keap1, Nrf2 is also repressed by ß-transducin repeat-containing protein (ß-TrCP), present in the Skp1-cullin-1-F-box protein (SCF) ubiquitin ligase complex SCFß-TrCP. The ability of SCFß-TrCP to suppress Nrf2 activity is itself enhanced by prior phosphorylation of the transcription factor by glycogen synthase kinase-3 (GSK-3) through formation of a DSGIS-containing phosphodegron. However, formation of the phosphodegron in Nrf2 by GSK-3 is inhibited by stimuli that activate protein kinase B (PKB)/Akt. In particular, PKB/Akt activity can be increased by phosphoinositide 3-kinase and mTORC2, thereby providing an explanation of why antioxidant-responsive element-driven genes are induced by growth factors and nutrients. Thus Nrf2 activity is tightly controlled via CRLKeap1 and SCFß-TrCP by oxidative stress and energy-based signals, allowing it to mediate adaptive responses that restore redox homeostasis and modulate intermediary metabolism. Based on the fact that Nrf2 influences multiple biochemical pathways in both positive and negative ways, it is likely its dose-response curve, in terms of susceptibility to certain degenerative disease, is U-shaped. Specifically, too little Nrf2 activity will lead to loss of cytoprotection, diminished antioxidant capacity, and lowered ß-oxidation of fatty acids, while conversely also exhibiting heightened sensitivity to ROS-based signaling that involves receptor tyrosine kinases and apoptosis signal-regulating kinase-1. By contrast, too much Nrf2 activity disturbs the homeostatic balance in favor of reduction, and so may have deleterious consequences including overproduction of reduced glutathione and NADPH, the blunting of ROS-based signal transduction, epithelial cell hyperplasia, and failure of certain cell types to differentiate correctly. We discuss the basis of a putative U-shaped Nrf2 dose-response curve in terms of potentially competing processes relevant to different stages of tumorigenesis.
Assuntos
Carcinogênese/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
Inactivation of the Ataxia Telangiectasia Mutated gene in chronic lymphocytic leukemia results in resistance to p53-dependent apoptosis and inferior responses to treatment with DNA damaging agents. Hence, p53-independent strategies are required to target Ataxia Telangiectasia Mutated-deficient chronic lymphocytic leukemia. As Ataxia Telangiectasia Mutated has been implicated in redox homeostasis, we investigated the effect of the Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia genotype on cellular responses to oxidative stress with a view to therapeutic targeting. We found that in comparison to Ataxia Telangiectasia Mutated-wild type chronic lymphocytic leukemia, pro-oxidant treatment of Ataxia Telangiectasia Mutated-null cells led to reduced binding of NF-E2 p45-related factor-2 to antioxidant response elements and thus decreased expression of target genes. Furthermore, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia cells contained lower levels of antioxidants and elevated mitochondrial reactive oxygen species. Consequently, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia, but not tumors with 11q deletion or TP53 mutations, exhibited differentially increased sensitivity to pro-oxidants both in vitro and in vivo. We found that cell death was mediated by a p53- and caspase-independent mechanism associated with apoptosis inducing factor activity. Together, these data suggest that defective redox-homeostasis represents an attractive therapeutic target for Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Homozigoto , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Mutação , Oxidantes/metabolismo , Fenótipo , Animais , Antioxidantes/metabolismo , Apoptose , Caspases/metabolismo , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta , Superóxidos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1(flox) allele and a rat CYP1A1-Cre transgene (Nrf1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Ácidos Graxos Dessaturases/genética , Regulação Enzimológica da Expressão Gênica , Metabolismo dos Lipídeos , Fator 1 Nuclear Respiratório/fisiologia , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Linhagem Celular , Cistina/metabolismo , Citocromo P-450 CYP1A1/genética , Ácidos Graxos Dessaturases/metabolismo , Feminino , Glutationa/metabolismo , Hepatócitos/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Ratos , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo , Elementos de Resposta , Ativação Transcricional , Triglicerídeos/metabolismoRESUMO
Induction of cytoprotective phaseâ 2 enzymes through inhibition of Keap1, a repressor of transcription factor Nrf2, is a cancer-prevention strategy. Compounds that elicit antiinflammatory and cytoprotective effects are promising candidates for chemoprevention. Novel analogues of 1-methyl-3-(2-oxopropylidene)indolin-2-one ('supercinnamaldehyde'; SCA) were synthesized, and their abilities to induce cytoprotective responses through Nrf2 induction and to suppress inflammatory responses were examined. 1-Methyl-3-(2-oxo-2-phenylethylidene)indolin-2-one (6) was identified as the lead compound. The compounds showed induction of Nrf2-dependent phaseâ 2 enzymes in Keap1+/+ mouse embryonic fibroblasts (MEFs), which was abrogated in Keap1-/- MEFs. The compounds also displayed a suppressive effect on NF-κB signaling that was at least partly responsible for inhibition of lipopolysaccharideinduced inflammatory responses. These SCA analogues exhibited cytoprotective and anti-inflammatory activities and may be developed further as chemopreventive agents.
Assuntos
Indóis/química , Fator 2 Relacionado a NF-E2/agonistas , NF-kappa B/antagonistas & inibidores , Acroleína/análogos & derivados , Acroleína/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Sobrevivência Celular , Citocinas/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HEK293 , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/prevenção & controleRESUMO
AIMS: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc(-) imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc(-) and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. RESULTS: PI3Ks induce system xc(-) through glycogen synthase kinase 3ß (GSK-3ß) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. INNOVATION: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3ß, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc(-). Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. CONCLUSION: PI3K-regulated system xc(-) activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate.