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1.
Hepatology ; 79(4): 829-843, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-37603610

RESUMO

BACKGROUND AND AIMS: Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. APPROACH AND RESULTS: To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. CONCLUSIONS: Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.


Assuntos
Fator 2 Relacionado a NF-E2 , Neoplasias , Animais , Humanos , Ratos , Proliferação de Células , Hepatócitos/metabolismo , Hiperplasia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Reprogramação Metabólica , Neoplasias/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Tri-Iodotironina/genética , Tri-Iodotironina/metabolismo
2.
Kidney Int ; 101(1): 92-105, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34767829

RESUMO

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.


Assuntos
Metabolismo dos Lipídeos , Voo Espacial , Animais , Pressão Sanguínea/genética , Calcificação Fisiológica , Expressão Gênica , Rim/metabolismo , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo
3.
Chem Res Toxicol ; 35(8): 1425-1432, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35862866

RESUMO

9,10-Phenanthrenequinone (9,10-PQ) is a toxicant in diesel exhaust particles and airborne particulate matter ≤2.5 µm in diameter. It is an efficient electron acceptor that readily reacts with dithiol compounds in vitro, resulting in the oxidation of thiol groups and concomitant generation of reactive oxygen species (ROS). However, it remains to be elucidated whether 9,10-PQ interacts with proximal protein dithiols. In the present study, we used thioredoxin 1 (Trx1) as a model of proteins with reactive proximal cysteines and examined whether it reacts with 9,10-PQ in cells and tissues, thereby affecting its catalytic activity and thiol status. Intratracheal injection of 9,10-PQ into mice resulted in protein oxidation and diminished Trx activity in the lungs. Using recombinant wild-type and C32S/C35S Trx1, we found that Cys32 and Cys35 selectively serve as electron donor sites for redox reactions with 9,10-PQ that lead to substantial inhibition of Trx activity. Addition of dithiothreitol restored the Trx activity inhibited by 9,10-PQ. Exposure of cultured cells to 9,10-PQ caused intracellular reactive oxygen species generation that led to protein oxidation, Trx1 dimerization, p38 phosphorylation, and apoptotic cell death. Overexpression of Trx1 blocked these 9,10-PQ-mediated events. These results suggest that the interaction of the reactive cysteines of Trx1 with 9,10-PQ causes oxidative stress, leading to disruption of redox homeostasis.


Assuntos
Elétrons , Tiorredoxinas , Animais , Cisteína/metabolismo , Homeostase , Camundongos , Oxidantes , Oxirredução , Fenantrenos , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo
4.
Genes Dev ; 28(6): 548-60, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24637114

RESUMO

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.


Assuntos
Relógios Circadianos/fisiologia , Regulação da Expressão Gênica/fisiologia , Glutationa/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Anticarcinógenos/farmacologia , Bleomicina/farmacologia , Relógios Circadianos/genética , Elementos E-Box/genética , Feminino , Homeostase , Isotiocianatos/farmacologia , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fibrose Pulmonar/induzido quimicamente , Sulfóxidos
5.
Am J Physiol Gastrointest Liver Physiol ; 321(4): G378-G388, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34405729

RESUMO

It was previously identified that systemic Nrf2 deletion attenuates pancreatic cancer progression in a mutant K-ras/p53-expressing mouse model (KPC mouse). In this study, the type of cell that is responsible for the retarded cancer progression was elucidated. Human pancreatic cancers were first examined, and elevated expression of NRF2-target gene products in α-smooth muscle actin-positive cells was found, suggesting that pancreatic stellate cells (PSCs) are involved in this process. Closer examination of primary cultured PSCs from Nrf2-deleted mice revealed that the cells were less proliferative and retained a lower migration capacity. The conditioned medium of Nrf2-deleted PSCs exhibited reduced growth-stimulating effects in pancreatic cancer cells. KPC mouse-derived pancreatic cancer cells coinjected with wild-type PSCs developed significantly larger subcutaneous tumors in immunodeficient mice than those coinjected with Nrf2-deleted PSCs. These results demonstrate that Nrf2 actively contributes to the function of PSCs to sustain KPC cancer progression, thus, suggesting that Nrf2 inhibition in PSCs may be therapeutically important in pancreatic cancer.NEW & NOTEWORTHY This study identified that Nrf2 contributes to PSC activation. Nrf2 deletion in PSCs resulted in attenuation of cancer-promoting role. Nrf2 in PSCs could be an attractive therapeutic target in pancreatic cancer.


Assuntos
Fator 2 Relacionado a NF-E2/genética , Neoplasias Pancreáticas/metabolismo , Células Estreladas do Pâncreas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética
6.
J Pharmacol Exp Ther ; 379(1): 33-40, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34321315

RESUMO

As the central regulator of the oxidative stress response, nuclear factor erythroid 2-related factor 2 (Nrf2) is attracting great interest as a therapeutic target for various cancers, and the possible clinical applications of novel Nrf2 inhibitors have been explored in Nrf2-activated cancers. In the present study, we specifically investigated halofuginone, which is derived from a natural plant alkaloid. We found that halofuginone administration decreased the number of pancreatic intraepithelial neoplasias in pancreas-specific Kras and p53 mutant (KPC) mice. In Nrf2-activated pancreatic cancer cell lines established from KPC mice, halofuginone rapidly depleted Nrf2 in Nrf2-activated cancer cells. Both in vitro and in vivo, it sensitized Nrf2-activated pancreatic cancer cells to gemcitabine, which is the first-line chemotherapy in clinical practice. In our mechanistic study, we found that halofuginone downregulated aldehyde dehydrogenase 3a1 (ALDH3A1) in mouse pancreatic cancer cells. The Nrf2 inducer diethyl maleate upregulated ALDH3A1, and knockdown of Aldh3a1 sensitized Nrf2-activated cancer cells to gemcitabine, strongly suggesting that ALDH3A1 is regulated by Nrf2 and that it contributes to gemcitabine resistance. The current study demonstrated the therapeutic benefits of halofuginone in Nrf2-activated pancreatic cancers. SIGNIFICANCE STATEMENT: We identified nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target aldehyde dehydrogenase 3a1 (ALDH3A1) as novel therapeutic targets in pancreatic cancer. They negatively affect the efficacy of a conventional chemotherapeutic agent, gemcitabine. We confirmed that Nrf2 plays a pivotal role in the induction of ALDH3A1.


Assuntos
Aldeído Desidrogenase/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Quinazolinonas/farmacologia , Quinazolinonas/uso terapêutico , Gencitabina
7.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672789

RESUMO

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras- and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras, i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras. These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.


Assuntos
Glutaminase/genética , Mutação , Fator 2 Relacionado a NF-E2/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Benzenoacetamidas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Malatos/farmacologia , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sulfetos/farmacologia , Tiadiazóis/farmacologia
8.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G419-G427, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961719

RESUMO

The activation of the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) pathway contributes to cancer progression in addition to oxidative stress responses. Loss-of-function Keap1 mutations were reported to activate Nrf2, leading to cancer progression. We examined the effects of Keap1 deletion in a cholangiocarcinoma mouse model using a mutant K-ras/p53 mouse. Introduction of the Keap1 deletion into liver-specific mutant K-ras/p53 expression resulted in the formation of invasive cholangiocarcinoma. Comprehensive analyses of the gene expression profiles identified broad upregulation of Nrf2-target genes such as Nqo1 and Gstm1 in the Keap1-deleted mutant K-ras/p53 expressing livers, accompanied by upregulation of cholangiocyte-related genes. Among these genes, the transcriptional factor Sox9 was highly expressed in the dysplastic bile duct. The Keap-Nrf2-Sox9 axis might serve as a novel therapeutic target for cholangiocarcinoma.NEW & NOTEWORTHY The Keap1-Nrf2 system has a wide variety of effects in addition to the oxidative stress response in cancer cells. Addition of the liver-specific Keap1 deletion to mice harboring mutant K-ras and p53 accelerated cholangiocarcinoma formation, together with the hallmarks of Nrf2 activation. This process involved the expansion of Sox9-positive cells, indicating increased differentiation toward the cholangiocyte phenotype.


Assuntos
Neoplasias dos Ductos Biliares/genética , Transformação Celular Neoplásica/genética , Colangiocarcinoma/genética , Deleção de Genes , Genes ras , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Mutação , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Diferenciação Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/deficiência , Masculino , Camundongos Transgênicos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Invasividade Neoplásica , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcriptoma , Proteína Supressora de Tumor p53/metabolismo
9.
Genes Cells ; 23(5): 386-392, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29542224

RESUMO

Keap1 is a negative regulator of Nrf2, a master transcription factor that regulates cytoprotection against oxidative and electrophilic stresses. Although several studies have suggested that the Keap1-Nrf2 system contributes to bone formation besides the maintenance of redox homeostasis, how Nrf2 hyperactivation by Keap1 deficiency affects the bone formation remains to be explored, as the Keap1-null mice are juvenile lethal. To overcome this problem, we used viable Keap1-deficient mice that we have generated by deleting the esophageal Nrf2 in Keap1-null mice (NEKO mice). We found that the NEKO mice exhibit small body size and low bone density. Although nephrogenic diabetes insipidus has been observed in both the NEKO mice and renal-specific Keap1-deficient mice, the skeletal phenotypes are not recapitulated in the renal-specific Keap1-deficient mice, suggesting that the skeletal phenotype by Nrf2 hyperactivation is not related to the renal phenotype. Experiments with primary culture cells derived from Keap1-null mice showed that differentiation of both osteoclasts and osteoblasts was attenuated, showing that impaired differentiation of osteoblasts rather than osteoclasts is responsible for bone hypoplasia caused by Nrf2 hyperactivation. Thus, we propose that the appropriate control of Nrf2 activity by Keap1 is essential for maintaining bone homeostasis.


Assuntos
Doenças Ósseas/etiologia , Diferenciação Celular , Regulação da Expressão Gênica , Fator 2 Relacionado a NF-E2/fisiologia , Osteoblastos/patologia , Osteoclastos/patologia , Animais , Doenças Ósseas/patologia , Células Cultivadas , Feminino , Homeostase , Proteína 1 Associada a ECH Semelhante a Kelch/fisiologia , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese
10.
J Hepatol ; 69(3): 635-643, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29758334

RESUMO

BACKGROUND & AIMS: Dysregulation of the Keap1-Nrf2 pathway has been observed in experimental and human tumors, suggesting possible roles of the pathway in cancer development. Herein, we examined whether Nrf2 (Nfe2l2) activation occurs at early steps of rat hepatocarcinogenesis, to assess critical contributions of Nrf2 to the onset of hepatocellular carcinoma (HCC). METHODS: We used wild-type (WT) and Nrf2 knockout (Nrf2KO) rats treated with a single injection of diethylnitrosamine (DENA) followed by choline-devoid methionine-deficient (CMD) diet. This experimental model causes massive fatty liver and steatohepatitis with fibrosis and enables identification of early stages of hepatocarcinogenesis. RESULTS: We found that Nrf2 activation takes place in early preneoplastic lesions identified by the marker glutathione S-transferase placental form (GSTP). Nrf2 missense mutations, known to disrupt the Keap1-Nrf2 binding, were present in 65.7% of GSTP-positive foci. Nrf2KO rats were used to directly investigate whether Nrf2 is critical for initiation and/or clonal expansion of DENA-damaged hepatocytes. While Nrf2 genetic inactivation did not alter DENA-induced initiation, it led to increased liver injury and chronic compensatory hepatocyte regeneration when rats were fed a CMD diet. However, in spite of such a permissive environment, the livers of Nrf2KO rats did not display any preneoplastic lesion unlike those of WT rats. CONCLUSIONS: These results demonstrate that, in a model of hepatocarcinogenesis resembling human non-alcoholic fatty liver disease: i) Nrf2 is activated at early steps of the tumorigenic process and ii) Nrf2 is mandatory for the clonal expansion of initiated cells, indicating that Nrf2 is critical in the onset of HCC. LAY SUMMARY: Dysregulation of the Keap1-Nrf2 molecular pathway has been observed in human tumors. In a nutritional model of hepatocarcinogenesis, the protein Nrf2 is frequently mutated/activated at early steps of the tumorigenic process. Herein, we show that Nrf2 is mandatory for the development of preneoplastic lesions. These results suggest that Nrf2 has a critical role in the onset of hepatocellular carcinoma.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular , Colina/farmacologia , Neoplasias Hepáticas , Metionina/farmacologia , Fator 2 Relacionado a NF-E2 , Alquilantes/farmacologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Dieta/métodos , Dietilnitrosamina/farmacologia , Modelos Animais de Doenças , Inativação Gênica , Lipotrópicos/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Ratos , Resultado do Tratamento
11.
Am J Physiol Gastrointest Liver Physiol ; 314(1): G65-G74, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28971839

RESUMO

The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system has a wide variety of effects in addition to the oxidative stress response, such as growth promotion and chemoresistance of cancer cells. Nrf2 is constitutively activated in most cancer cells. However, the activation of Nrf2 together with oncogenic mutations does not always result in cancer promotion. K-rasLSL-G12D/+:: p53LSL-R172H/+:: Pdx-1-Cre (KPC) mice are an established model of pancreatic cancer that specifically express mutants of both K-ras and p53 in the pancreas by using Pdx-1-Cre. We here generated Pdx-1-Cre::K-rasLSL-G12D/+:: Keap1fl/fl (KC::Keap1) and KPC:: Keap1fl/fl (KPC::Keap1) mice in which Nrf2 is constitutively activated by Keap1 deletion. KC::Keap1 and KPC::Keap1 mice started to die or showed obvious weakness at approximately around 40 days after birth. Histological examination revealed that KC::Keap1 and KPC::Keap1 mice did not develop pancreatic cancer but, instead, progressive atrophy of the pancreatic parenchyma. In these mice, amylase-positive acinar cells as well as insulin- and glucagon-positive islet cells were decreased and surrounded by fibrotic tissues. KC::Keap1 and KPC::Keap1 mice presented lower body weight and glucose levels than C::Keap1 mice, presumably resulting from pancreatic exocrine insufficiency. Histological changes were not obvious in C::Keap1 and PC::Keap1 mice. The presence of the p53 mutation did not affect the phenotypes in KC::Keap1 mice. Heterologous or homologous Nrf2 deletion ( Nrf2+/- or Nrf2-/-) rescued the pancreatic phenotypes, weight loss, and hypoglycemia in KC::Keap1 mice, suggesting that Nrf2 is a major downstream target of Keap1. In conclusion, simultaneous K-ras activation and Keap1 deletion caused progressive atrophy of the pancreatic parenchyma in mice. NEW & NOTEWORTHY Aberrant activation of the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system usually promotes carcinogenesis, and we assumed that simultaneous activation of K-ras and Nrf2 might promote pancreatic carcinogenesis. Conditional expression of mutant K-ras and Keap1 deletion did not result in pancreatic cancer development. Instead, these mice developed progressive loss of pancreatic parenchyma, accompanied by body weight loss and hypoglycemia, presumably because of pancreatic exocrine insufficiency. Nrf2 activation by Keap1 deletion concomitant with K-ras activation cause pancreatic atrophy.


Assuntos
Deleção de Genes , Genes ras , Proteína 1 Associada a ECH Semelhante a Kelch/deficiência , Pâncreas/metabolismo , Pancreatite Crônica/metabolismo , Animais , Atrofia , Glicemia/metabolismo , Modelos Animais de Doenças , Feminino , Genes p53 , Predisposição Genética para Doença , Glucagon/sangue , Proteínas de Homeodomínio/genética , Insulina/sangue , Integrases/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Masculino , Camundongos Knockout , Mutação , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Pâncreas/patologia , Pancreatite Crônica/genética , Pancreatite Crônica/patologia , Tecido Parenquimatoso/metabolismo , Tecido Parenquimatoso/patologia , Fenótipo , Transativadores/genética
12.
Carcinogenesis ; 38(6): 661-670, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29240881

RESUMO

The Keap1-Nrf2 system contributes to the maintenance of homeostasis by regulating oxidative stress responses in normal tissues and organs, and is exploited in various cancers for proliferation, survival and acquisition of therapy resistance. Pancreatic cancer remains one of the intractable cancers, despite the improved clinical outcomes of other types of cancer, due to its invasive and refractory nature to therapeutic intervention. The current study aimed to clarify the contribution of Nrf2 to pancreatic carcinogenesis using a pancreas-specific mutant K-ras and p53 (KPC) mouse model. Deletion of Nrf2 in KPC mice (KPCN) decreased the formation of precancerous lesions as well as the development of invasive pancreatic cancer. The pancreatic tumor-derived cancer cell lines from KPCN mouse showed decreased expression of glutathione S-transferases (GST), UDP glucuronosyltransferases (UGT) and ABC transporters. Along with these biochemical changes, cell lines from KPCN mice revealed increased sensitivity to oxidative stress and chemotherapeutic agent. The current study revealed that Nrf2 contributes to pancreatic carcinogenesis in a way distinct from the chemoresistance of lung and esophagus, and that Nrf2 could be a novel therapeutic target of pancreatic cancer.


Assuntos
Genes p53 , Mutação , Fator 2 Relacionado a NF-E2/fisiologia , Neoplasias Pancreáticas/etiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Masculino , Camundongos , Estresse Oxidativo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Lesões Pré-Cancerosas/etiologia , Gencitabina
13.
J Biol Chem ; 291(4): 1826-1840, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26620562

RESUMO

The transcription factor Bach2 regulates the immune system at multiple points, including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells are still unclear. Here, we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of the B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in the mature B cell line BAL17. Among the sites identified, serine 535 (Ser-535) was critical for the regulation of Bach2 because a single mutation of Ser-535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas Ser-509 played an auxiliary role. Bach2 repressor activity was enhanced by the Ser-535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells.


Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Núcleo Celular/metabolismo , Motivos de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Núcleo Celular/genética , Células Cultivadas , Feminino , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Células Precursoras de Linfócitos B/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
15.
Toxicol Appl Pharmacol ; 295: 37-46, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26827822

RESUMO

Cadmium is an environmental electrophile that modifies protein reactive thiols such as Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear factor-erythroid 2-related factor 2 (Nrf2). In the present study, we investigated a role of the Keap1-Nrf2 system in cellular response to cadmium in vascular endothelial cells. Exposure of bovine aortic endothelial cells to cadmium resulted in modification of Keap1 and Nrf2 activation, thereby up-regulating not only its typical downstream proteins but also metallothionein-1/2. Experiments with siRNA-mediated knockdown of Nrf2 or Keap1 supported participation of the Keap1-Nrf2 system in the modulation of metallothionein-1/2 expression. Furthermore, chromatin immunoprecipitation assay showed that Nrf2 was recruited to the antioxidant response element of the promoter region of the bovine metallothionein-2 gene in the presence of cadmium. These results suggest that the transcription factor Nrf2 plays, at least in part, a role in the changes in metallothionein expression mediated by exposure to cadmium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Cádmio/farmacologia , Proteínas do Citoesqueleto/biossíntese , Células Endoteliais/metabolismo , Metalotioneína/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Animais , Elementos de Resposta Antioxidante/efeitos dos fármacos , Bovinos , Técnicas de Cultura de Células , Cromatografia Líquida , Relação Dose-Resposta a Droga , Proteína 1 Associada a ECH Semelhante a Kelch , Espectrometria de Massas , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos
16.
Hepatology ; 59(6): 2371-82, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24443206

RESUMO

UNLABELLED: Hepatectomy is a standard therapy that allows liver cancer patients to achieve long-term survival. Preceding hepatectomy, portal vein embolization (PVE) is frequently performed to increase the remnant liver size and reduce complications. Although the clinical importance of PVE is widely accepted, molecular mechanisms by which PVE leads to compensatory hypertrophy of nonembolized lobes remain elusive. We hypothesized that NF-E2-related factor 2 (Nrf2), a master regulator of cytoprotection, promotes compensatory liver hypertrophy after PVE. To address this hypothesis, we utilized three mouse lines and the portal vein branch ligation (PVBL) technique, which primarily induces the redistribution of the portal bloodstream in liver in a manner similar to PVE. PVBL was conducted in Kelch-like ECH-associated protein 1 (Keap1) conditional knockout (Keap1-CKO) mice in which Nrf2 is constitutively activated, along with Nrf2-deficient (Nrf2-KO) mice. We found that hypertrophy of nonligated lobes after PVBL was enhanced and limited in Keap1-CKO and Nrf2-KO mice, respectively, compared to wild-type mice. In Keap1-CKO mice, Nrf2 activity was increased, consistent with transient activation of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, and reactive hepatocyte proliferation was significantly prolonged after PVBL. Importantly, Nrf2 activation by a chemical inducer was also effective for enhancement of hypertrophy after PVBL. CONCLUSION: Nrf2 supports compensatory liver hypertrophy after PVBL. This finding is particularly intriguing, because the primary effect of PVBL is limited to the alteration of bloodstream; this effect is much milder than changes resulting from hepatectomy, in which intrahepatic bloodstream and bile production cease. Our results suggest that premedication with an Nrf2 inducer may be a promising strategy to improve the outcome of PVE; this approach expands the indication of hepatectomy to patients with poorer liver function.


Assuntos
Fígado/fisiologia , Fator 2 Relacionado a NF-E2/fisiologia , Veia Porta , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Proteínas do Citoesqueleto/genética , Embolização Terapêutica , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatectomia , Hipertrofia , Imidazóis , Proteína 1 Associada a ECH Semelhante a Kelch , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido Oleanólico/análogos & derivados , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
17.
Proc Natl Acad Sci U S A ; 109(34): 13561-6, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22872865

RESUMO

The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system is essential for cytoprotection against oxidative and electrophilic insults. Under unstressed conditions, Keap1 serves as an adaptor for ubiquitin E3 ligase and promotes proteasomal degradation of Nrf2, but Nrf2 is stabilized when Keap1 is inactivated under oxidative/electrophilic stress conditions. Autophagy-deficient mice show aberrant accumulation of p62, a multifunctional scaffold protein, and develop severe liver damage. The p62 accumulation disrupts the Keap1-Nrf2 association and provokes Nrf2 stabilization and accumulation. However, individual contributions of p62 and Nrf2 to the autophagy-deficiency-driven liver pathogenesis have not been clarified. To examine whether Nrf2 caused the liver injury independent of p62, we crossed liver-specific Atg7::Keap1-Alb double-mutant mice into p62- and Nrf2-null backgrounds. Although Atg7::Keap1-Alb::p62(-/-) triple-mutant mice displayed defective autophagy accompanied by the robust accumulation of Nrf2 and severe liver injury, Atg7::Keap1-Alb::Nrf2(-/-) triple-mutant mice did not show any signs of such hepatocellular damage. Importantly, in this study we noticed that Keap1 accumulated in the Atg7- or p62-deficient mouse livers and the Keap1 level did not change by a proteasome inhibitor, indicating that the Keap1 protein is constitutively degraded through the autophagy pathway. This finding is in clear contrast to the Nrf2 degradation through the proteasome pathway. We also found that treatment of cells with tert-butylhydroquinone accelerated the Keap1 degradation. These results thus indicate that Nrf2 accumulation is the dominant cause to provoke the liver damage in the autophagy-deficient mice. The autophagy pathway maintains the integrity of the Keap1-Nrf2 system for the normal liver function by governing the Keap1 turnover.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Enzimas de Conjugação de Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Autofagia , Proteína 7 Relacionada à Autofagia , Proteínas do Citoesqueleto/química , Células Hep G2 , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína 1 Associada a ECH Semelhante a Kelch , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição TFIIH , Fatores de Transcrição/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo
18.
Nephrol Dial Transplant ; 29(4): 783-91, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24523358

RESUMO

BACKGROUND: NFE2-related factor 2 (Nrf2) is a master regulatory transcription factor for antioxidant genes. Inhibition of its adaptor protein, Kelch-like ECH-associated protein 1 (Keap1), activates Nrf2. Podocyte injury triggers the progressive deterioration of glomerular damage toward glomerulosclerosis. We examined whether modulation of the Keap1-Nrf2 system has an impact on this process. METHODS: Nrf2 null-mutant (KO) and Keap1 hypomorphic knockdown (KD) mice were crossed with NEP25 mice, in which podocyte-specific injury can be induced by an immunotoxin. RESULTS: Thiobarbituric acid reactive substances, 8-hydroxydeoxyguanosine and phosphorylated JNK were increased in the injured NEP25 kidney. Real-time PCR revealed that Keap1 KD upregulated Nrf2 target genes, including Gclc, Gclm, Gstp1, Gstp2 and Nqo1 in the glomerulus. However, podocyte injury did not upregulate these genes in Keap1 wild-type mice, nor did it further increase the expression of those genes in Keap1 KD mice. Three weeks after the induction of podocyte injury, glomerulosclerosis was considerably more attenuated in Keap1 KD mice than in control mice (median sclerosis index, 0.27 versus 3.03, on a 0-4 scale). Keap1 KD mice also showed considerably preserved nephrin staining (median index, 6.76 versus 0.91, on a 0-8 scale) and decreased glomeruli containing desmin-positive injured podocytes (median percentage, 24.5% versus 85.8%), along with a decrease in mRNAs for Fn1, Tgfb1, Col4a4 and Col1a2. CONCLUSIONS: Thus, podocyte injury cannot effectively activate Nrf2, but Nrf2 activation by Keap1 knockdown attenuates glomerulosclerosis. These results indicate that the Nrf2-Keap1 system is a promising drug target for the treatment of chronic kidney diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Anticorpos Monoclonais/toxicidade , Proteínas do Citoesqueleto/genética , DNA/genética , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/genética , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Alelos , Animais , Antioxidantes/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/biossíntese , Modelos Animais de Doenças , Exotoxinas/toxicidade , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Imunotoxinas , Proteína 1 Associada a ECH Semelhante a Kelch , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Mutantes , Fator 2 Relacionado a NF-E2/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Reação em Cadeia da Polimerase em Tempo Real
19.
Cell Stress Chaperones ; 29(3): 497-509, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38763404

RESUMO

Bcl2-associated athanogene-1 protein (Bag1) acts as a co-chaperone of heat shock protein 70 and heat shock cognate 70 and regulates multiple cellular processes, including cell proliferation, apoptosis, environmental stress response, and drug resistance. Since Bag1 knockout mice exhibited fetal lethality, the in vivo function of Bag1 remains unclear. In this study, we established a mouse line expressing Bag1 gene missing exon 5, which corresponds to an encoding region for the interface of heat shock protein 70/heat shock cognate 70. Despite mice carrying homoalleles of the Bag1 mutant (Bag1Δex5) expressing undetectable levels of Bag1, Bag1Δex5 homozygous mice developed without abnormalities. Bag1Δex5 protein was found to be highly unstable in cells and in vitro. We found that the growth of mouse embryonic fibroblasts derived from Bag1Δex5-homo mice was attenuated by doxorubicin and a glutathione (GSH) synthesis inhibitor, buthionine sulfoximine. In response to buthionine sulfoximine, Bag1Δex5-mouse embryonic fibroblasts exhibited a higher dropping rate of GSH relative to the oxidized glutathione level. In addition, Bag1 might mitigate cellular hydrogen peroxide levels. Taken together, our results demonstrate that the loss of Bag1 did not affect mouse development and that Bag1 is involved in intracellular GSH homeostasis, namely redox homeostasis.


Assuntos
Proteínas de Ligação a DNA , Fibroblastos , Glutationa , Fatores de Transcrição , Animais , Fibroblastos/metabolismo , Glutationa/metabolismo , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Doxorrubicina/farmacologia , Butionina Sulfoximina/farmacologia , Embrião de Mamíferos/metabolismo , Proliferação de Células , Camundongos Knockout , Peróxido de Hidrogênio/metabolismo
20.
J Biochem ; 175(6): 611-627, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38268329

RESUMO

Whole blood transcriptome analysis is a valuable approachin medical research, primarily due to the ease of sample collection and the richness of the information obtained. Since the expression profile of individual genes in the analysis is influenced by medical traits and demographic attributes such as age and gender, there has been a growing demand for a comprehensive database for blood transcriptome analysis. Here, we performed whole blood RNA sequencing (RNA-seq) analysis on 576 participants stratified by age (20-30s and 60-70s) and gender from cohorts of the Tohoku Medical Megabank (TMM). A part of female segment included pregnant women. We did not exclude the globin gene family in our RNA-seq study, which enabled us to identify instances of hereditary persistence of fetal hemoglobin based on the HBG1 and HBG2 expression information. Comparing stratified populations allowed us to identify groups of genes associated with age-related changes and gender differences. We also found that the immune response status, particularly measured by neutrophil-to-lymphocyte ratio (NLR), strongly influences the diversity of individual gene expression profiles in whole blood transcriptome analysis. This stratification has resulted in a data set that will be highly beneficial for future whole blood transcriptome analysis in the Japanese population.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Humanos , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Perfilação da Expressão Gênica/métodos , Japão , Idoso , Adulto Jovem , Fatores Etários , Fatores Sexuais , Povo Asiático/genética , População do Leste Asiático
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