Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443423

RESUMO

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-ß1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.


Assuntos
Cirrose Hepática Experimental/tratamento farmacológico , Melastomataceae/química , Neovascularização Patológica/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Citocinas/genética , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática Experimental/sangue , Neovascularização Patológica/sangue , Tamanho do Órgão/efeitos dos fármacos , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tioacetamida , Regulação para Cima/efeitos dos fármacos , Água , Perda de Peso/efeitos dos fármacos
2.
Mol Cell Biol ; 38(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29941490

RESUMO

Cancer cells often heavily depend on the ubiquitin-proteasome system (UPS) for their growth and survival. Irrespective of their strong dependence on the proteasome activity, cancer cells, except for multiple myeloma, are mostly resistant to proteasome inhibitors. A major cause of this resistance is the proteasome bounce-back response mediated by NRF1, a transcription factor that coordinately activates proteasome subunit genes. To identify new targets for efficient suppression of UPS, we explored, using immunoprecipitation and mass spectrometry, the possible existence of nuclear proteins that cooperate with NRF1 and identified O-linked N-acetylglucosamine transferase (OGT) and host cell factor C1 (HCF-1) as two proteins capable of forming a complex with NRF1. O-GlcNAcylation catalyzed by OGT was essential for NRF1 stabilization and consequent upregulation of proteasome subunit genes. Meta-analysis of breast and colorectal cancers revealed positive correlations in the relative protein abundance of OGT and proteasome subunits. OGT inhibition was effective at sensitizing cancer cells to a proteasome inhibitor both in culture cells and a xenograft mouse model. Since active O-GlcNAcylation is a feature of cancer metabolism, our study has clarified a novel linkage between cancer metabolism and UPS function and added a new regulatory axis to the regulation of the proteasome activity.


Assuntos
Fator 1 Relacionado a NF-E2/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Proteassoma/farmacologia , Acetilglucosamina/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Glicosilação , Células HEK293 , Células HeLa , Fator C1 de Célula Hospedeira/química , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Fator 1 Relacionado a NF-E2/química , Fator 1 Relacionado a NF-E2/genética , Neoplasias/genética , Fator 1 Nuclear Respiratório , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Contendo Repetições de beta-Transducina/química , Proteínas Contendo Repetições de beta-Transducina/metabolismo
3.
Molecules ; 23(4)2018 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-29641495

RESUMO

Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic conditions. Hypoxia-inducible factor-α (HIF-α) is a major transcription factor that orchestrates this protective effect. To activate HIF exogenously, without exposing cells to hypoxic conditions, many small-molecule inhibitors targeting prolyl hydroxylase domain-containing protein have been developed. In addition, suppression of factor inhibiting HIF-1 (FIH-1) has also been shown to have the potential to activate HIF-α. However, few small-molecule inhibitors of FIH-1 have been developed. In this study, we synthesized a series of furan- and thiophene-2-carbonyl amino acid derivatives having the potential to inhibit FIH-1. The inhibitory activities of these compounds were evaluated in SK-N-BE(2)c cells by measuring HIF response element (HRE) promoter activity. Several furan- and thiophene-2-carbonyl amino acid derivatives inhibited FIH-1 based on correlations among the docking score of the FIH-1 active site, the chemical structure of the compounds, and biological HIF-α/HRE transcriptional activity.


Assuntos
Furanos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oxigenases de Função Mista/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Tiofenos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Furanos/síntese química , Furanos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Oxigenases de Função Mista/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Repressoras/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiofenos/síntese química , Tiofenos/química , Ativação Transcricional/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 115(11): 2758-2763, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29472449

RESUMO

Nrf2 plays critical roles in animals' defense against electrophiles and oxidative stress by orchestrating the induction of cytoprotective genes. We previously isolated the zebrafish mutant it768, which displays up-regulated expression of Nrf2 target genes in an uninduced state. In this paper, we determine that the gene responsible for it768 was the zebrafish homolog of phosphomannomutase 2 (Pmm2), which is a key enzyme in the initial steps of N-glycosylation, and its mutation in humans leads to PMM2-CDG (congenital disorders of glycosylation), the most frequent type of CDG. The pmm2it768 larvae exhibited mild defects in N-glycosylation, indicating that the pmm2it768 mutation is a hypomorph, as in human PMM2-CDG patients. A gene expression analysis showed that pmm2it768 larvae display up-regulation of endoplasmic reticulum (ER) stress, suggesting that the activation of Nrf2 was induced by the ER stress. Indeed, the treatment with the ER stress-inducing compounds up-regulated the gstp1 expression in an Nrf2-dependent manner. Furthermore, the up-regulation of gstp1 by the pmm2 inactivation was diminished by knocking down or out double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), one of the main ER stress sensors, suggesting that Nrf2 was activated in response to the ER stress via the PERK pathway. ER stress-induced activation of Nrf2 was reported previously, but the results have been controversial. Our present study clearly demonstrated that ER stress can indeed activate Nrf2 and this regulation is evolutionarily conserved among vertebrates. Moreover, ER stress induced in pmm2it768 mutants was ameliorated by the treatment of the Nrf2-activator sulforaphane, indicating that Nrf2 plays significant roles in the reduction of ER stress.


Assuntos
Estresse do Retículo Endoplasmático , Fator 2 Relacionado a NF-E2/metabolismo , Fosfotransferases (Fosfomutases)/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Glicosilação , Mutação , Fator 2 Relacionado a NF-E2/genética , Fosfotransferases (Fosfomutases)/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Genes Cells ; 22(11): 939-952, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29044949

RESUMO

Erythropoietin (EPO) is a hormone that promotes proliferation, differentiation and survival of erythroid progenitors. EPO gene expression is regulated in a tissue-specific and hypoxia-inducible manner and is mainly restricted to renal EPO-producing cells after birth. Chronic kidney disease (CKD) confers high risk for renal anemia due to lower EPO production from injured kidneys. In transgenic reporter lines of mice, disruption of a GATA-binding motif within the Epo gene promoter-proximal region restores constitutive reporter expression in epithelial cells. Here, mitoxantrone and its analogues, identified as GATA factor inhibitors through high-throughput chemical library screenings, markedly induce EPO/Epo gene expression in epithelium-derived cell lines and mice regardless of oxygen levels. In contrast, mitoxantrone interferes with hypoxia-induced EPO gene expression in Hep3B cells. Cryptic promoters are created for the EPO/Epo gene expression in epithelial cells upon mitoxantrone treatment, and consequently, unique 5'-untranslated regions are generated. The mitoxantrone-induced aberrant transcripts contribute to the reporter protein production in epithelial cells that carry the reporter gene in the proper reading frame of mouse Epo gene. Thus, EPO production in uninjured adult epithelial cells may be a therapeutic approach for renal anemia in patients with CKD.


Assuntos
Células Epiteliais/metabolismo , Eritropoetina/metabolismo , Fatores de Transcrição GATA/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Mitoxantrona/farmacologia , Insuficiência Renal Crônica/metabolismo , Anemia/tratamento farmacológico , Anemia/metabolismo , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Eritropoetina/antagonistas & inibidores , Eritropoetina/genética , Fatores de Transcrição GATA/metabolismo , Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitoxantrona/química , Regiões Promotoras Genéticas , Insuficiência Renal Crônica/patologia , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia
6.
Anal Sci ; 33(9): 989-991, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28890499

RESUMO

Luminescent europium-doped layered titanates (Eu-TiOx) were synthesized and complexed with horseradish peroxidase (HRP) and glucose oxidase (GOx). The emission of a resultant Eu-TiOx/HRP/GOx complex decreased upon the addition of glucose in the presence of guaiacol. The emission decrease was dependent on the concentrations of glucose, and the detection limit for glucose was 3.1 µM. The proposed system would be promising as a new detection method for glucose.


Assuntos
Técnicas Biossensoriais , Európio/química , Glucose Oxidase/metabolismo , Glucose/análise , Peroxidase do Rábano Silvestre/metabolismo , Titânio/química , Európio/metabolismo , Fluorescência , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Espectrometria de Fluorescência , Fatores de Tempo , Titânio/metabolismo
7.
Biochem Biophys Res Commun ; 489(1): 21-28, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28533088

RESUMO

CYP11B2 is a key enzyme involved in the synthesis of the mineralocorticoid aldosterone. CYP11B2 expression in the adrenal glands is controlled by the renin-angiotensin system (RAS), and plays an important role in the maintenance of electrolyte metabolism in higher organisms. Abnormal overexpression of CYP11B2 results in the disruption of mineral balance and can lead to hypertension. Though the molecular mechanism of the regulation of CYP11B2 expression has remained elusive, we hypothesize that compounds that prevent CYP11B2 expression could represent a novel class of antihypertensive drugs. In this study, we established a high-throughput screening system to identify such compounds, and subsequently carried out chemical screening. We found that the ubiquitin-proteasome inhibitor bortezomib could suppress CYP11B2 expression and secretion of aldosterone induced by angiotensin II (Ang II) in adrenocortical H295R cells. Moreover, bortezomib down-regulated the Cyp11b2 mRNA expression facilitated in the adrenal gland of Tsukuba hypertensive mice, resulting in subsequent lowering of their blood pressures. Furthermore, we observed the characteristic alteration of H3K27ac in the adrenal CYP11B2 gene promoter induced by Ang II stimulation, which was found to be disrupted by bortezomib. Taken together, these results suggest the possibility of developing novel antihypertensive drugs that prevent CYP11B2 expression.


Assuntos
Aldosterona/biossíntese , Bortezomib/farmacologia , Citocromo P-450 CYP11B2/biossíntese , Citocromo P-450 CYP11B2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Bortezomib/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP11B2/genética , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Molecular , Relação Estrutura-Atividade
8.
Free Radic Biol Med ; 103: 236-247, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28039084

RESUMO

The KEAP1-NRF2 system regulates the cellular defence against oxidative and xenobiotic stresses. NRF2 is a transcription factor that activates the expression of cytoprotective genes encoding antioxidative, detoxifying and metabolic enzymes as well as transporters. Under normal conditions, KEAP1 represses NRF2 activity by degrading the NRF2 protein. When cells are exposed to stresses, KEAP1 stops promoting NRF2 degradation, and NRF2 rapidly accumulates and activates the transcription of target genes. Constitutive accumulation of NRF2 via a variety of mechanisms that disrupt KEAP1-mediated NRF2 degradation has been observed in various cancer types. Constitutive NRF2 accumulation confers cancer cells with a proliferative advantage as well as resistance to anti-cancer drugs and radiotherapies. To suppress the chemo- and radio-resistance of cancer cells caused by NRF2 accumulation, we conducted high-throughput chemical library screening for NRF2 inhibitors and identified febrifugine derivatives. We found that application of the less-toxic derivative halofuginone in a low dose range rapidly reduced NRF2 protein levels. Halofuginone induced a cellular amino acid starvation response that repressed global protein synthesis and rapidly depleted NRF2. Halofuginone treatment ameliorated the resistance of NRF2-addicted cancer cells to anti-cancer drugs both in vitro and in vivo. These results provide preclinical proof-of-concept evidence for halofuginone as an NRF2 inhibitor applicable to treatment of chemo- and radio-resistant forms of cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Células A549 , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Expressão Gênica , Humanos , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Quinazolinonas/administração & dosagem , Quinazolinonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Mol Cell Biol ; 37(4)2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27920251

RESUMO

Eukaryotic cells maintain protein homeostasis through the activity of multiple basal and inducible systems, which function in concert to allow cells to adapt to a wide range of environmental conditions. Although the transcriptional programs regulating individual pathways have been studied in detail, it is not known how the different pathways are transcriptionally integrated such that a deficiency in one pathway can be compensated by a change in an auxiliary response. One such pathway that plays an essential role in many proteostasis responses is the ubiquitin-proteasome system, which functions to degrade damaged, unfolded, or short half-life proteins. Transcriptional regulation of the proteasome is mediated by the transcription factor Nrf1. Using a conditional knockout mouse model, we found that Nrf1 regulates protein homeostasis in the endoplasmic reticulum (ER) through transcriptional regulation of the ER stress sensor ATF6. In Nrf1 conditional-knockout mice, a reduction in proteasome activity is accompanied by an ATF6-dependent downregulation of the endoplasmic reticulum-associated degradation machinery, which reduces the substrate burden on the proteasome. This indicates that Nrf1 regulates a homeostatic shift through which proteostasis in the endoplasmic reticulum and cytoplasm are coregulated based on a cell's ability to degrade proteins.


Assuntos
Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Homeostase , Transcrição Genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina , Regulação para Baixo/genética , Elementos Facilitadores Genéticos/genética , Homeostase/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Modelos Biológicos , Fator 1 Nuclear Respiratório/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo
11.
Genes Cells ; 20(7): 563-77, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25940588

RESUMO

NRF1 (NF-E2-p45-related factor 1) plays an important role in the regulation of genes encoding proteasome subunits, a cystine transporter, and lipid-metabolizing enzymes. Global and tissue-specific disruptions of the Nrf1 gene in mice result in embryonic lethality and spontaneous development of severe tissue defects, respectively, suggesting NRF1 plays a critical role in vivo. Mechanistically, the continuous degradation of the NRF1 protein by the proteasome is regarded as a major regulatory nexus of NRF1 activity. To develop NRF1-specific inducers that act to overcome the phenotypes related to the lack of NRF1 activity, we constructed a novel NRF1ΔC-Luc fusion protein reporter and developed cell lines that stably express the reporter in Hepa1c1c7 cells for use in high-throughput screening. In screening of a chemical library with this reporter system, we identified two hit compounds that significantly induced luciferase activity. Through an examination of a series of derivatives of one of the hit compounds, we identified T1-20, which induced a 70-fold increase in luciferase activity. T1-20 significantly increased the level of NRF1 protein in the mouse liver, indicating that the compound is also functional in vivo. Thus, these results show the successful identification of the first small chemical compounds which specifically and significantly induce NRF1.


Assuntos
Bases de Dados de Compostos Químicos , Descoberta de Drogas , Fator 1 Nuclear Respiratório/química , Fator 1 Nuclear Respiratório/metabolismo , Compostos Orgânicos/metabolismo , Animais , Linhagem Celular Tumoral , Vetores Genéticos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Fígado/metabolismo , Camundongos
12.
Mol Cell Biol ; 35(15): 2658-72, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26012551

RESUMO

Erythropoietin (Epo) is produced in the kidney and liver in a hypoxia-inducible manner via the activation of hypoxia-inducible transcription factors (HIFs) to maintain oxygen homeostasis. Accelerating Epo production in hepatocytes is one plausible therapeutic strategy for treating anemia caused by kidney diseases. To elucidate the regulatory mechanisms of hepatic Epo production, we analyzed mouse lines harboring liver-specific deletions of genes encoding HIF-prolyl-hydroxylase isoforms (PHD1, PHD2, and PHD3) that mediate the inactivation of HIF1α and HIF2α under normal oxygen conditions. The loss of all PHD isoforms results in both polycythemia, which is caused by Epo overproduction, and fatty livers. We found that deleting any combination of two PHD isoforms induces polycythemia without steatosis complications, whereas the deletion of a single isoform induces no apparent phenotype. Polycythemia is prevented by the loss of either HIF2α or the hepatocyte-specific Epo gene enhancer (EpoHE). Chromatin analyses show that the histones around EpoHE dissociate from the nucleosome structure after HIF2α activation. HIF2α also induces the expression of HIF3α, which is involved in the attenuation of Epo production. These results demonstrate that the total amount of PHD activity is more important than the specific function of each isoform for hepatic Epo expression regulated by a PHD-HIF2α-EpoHE cascade in vivo.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Eritropoetina/biossíntese , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Anemia/etiologia , Animais , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Fígado Gorduroso/genética , Hepatócitos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Policitemia/genética , Proteínas Repressoras , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Genética/genética
13.
Tohoku J Exp Med ; 235(2): 151-9, 2015 02.
Artigo em Inglês | MEDLINE | ID: mdl-25746387

RESUMO

The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.


Assuntos
Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Animais , Sequência de Bases , Hipóxia Celular/genética , Linhagem Celular , Humanos , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Ratos , Elementos de Resposta/genética , Estresse Fisiológico/genética
14.
Nat Med ; 21(3): 281-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25706873

RESUMO

Metabolic reprogramming occurs in response to the cellular environment to mediate differentiation, but the fundamental mechanisms linking metabolic processes to differentiation programs remain to be elucidated. During osteoclast differentiation, a shift toward more oxidative metabolic processes occurs. In this study we identified the de novo DNA methyltransferase 3a (Dnmt3a) as a transcription factor that couples these metabolic changes to osteoclast differentiation. We also found that receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclastogenesis, induces this metabolic shift towards oxidative metabolism, which is accompanied by an increase in S-adenosylmethionine (SAM) production. We found that SAM-mediated DNA methylation by Dnmt3a regulates osteoclastogenesis via epigenetic repression of anti-osteoclastogenic genes. The importance of Dnmt3a in bone homeostasis was underscored by the observations that Dnmt3a-deficient osteoclast precursor cells do not differentiate efficiently into osteoclasts and that mice with an osteoclast-specific deficiency in Dnmt3a have elevated bone mass due to a smaller number of osteoclasts. Furthermore, inhibition of DNA methylation by theaflavin-3,3'-digallate abrogated bone loss in models of osteoporosis. Thus, this study reveals the role of epigenetic processes in the regulation of cellular metabolism and differentiation, which may provide the molecular basis for a new therapeutic strategy for a variety of bone disorders.


Assuntos
Reabsorção Óssea/genética , Diferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Osteoclastos/citologia , Osteogênese/genética , Ligante RANK/genética , S-Adenosilmetionina/metabolismo , Animais , Biflavonoides/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA/genética , Regulação para Baixo , Expressão Gênica , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ligante RANK/fisiologia , Fatores de Transcrição
15.
Mol Cell Biol ; 34(20): 3800-16, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25092871

RESUMO

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/metabolismo
16.
Genes Cells ; 19(8): 650-65, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25041126

RESUMO

Nrf1 (NF-E2-related factor 1) is a basic region leucine zipper-type transcription factor belonging to the CNC (cap-'n'-collar) family. Major pathophysiological contribution of Nrf1 remains unclear. As single nucleotide polymorphism rs3764400 in 5'-flanking region of NRF1 gene appears to associate with obesity, in this study, we focused on the Nrf1 function on metabolism. We found that the risk C allele of rs3764400 increased NRF1 gene transcriptional activity compared with the T allele in hepatoma cell lines. Therefore, we newly established Nrf1 transgenic (Nrf1-Tg) mouse lines and examined roles that Nrf1 plays on the obesity and metabolism. Unexpectedly, Nrf1 over-expression repressed bodyweight gain in both lean and diet-induced obesity mice. Of note, Nrf1-Tg mice showed rise in blood glucose levels; Nrf1 strongly reduced glucose infusion rate in euglycemic-hyperinsulinemic clamp test and increased blood glucose levels in insulin tolerance test, indicating that Nrf1 induces insulin resistance in mice. Nrf1 repressed insulin-regulated glycolysis-related gene expression and gave rise to loss of glucose-6-phosphate and fructose-6-phosphate contents in liver. Consistently, Nrf1 heterozygote improved impaired glucose regulations in diet-induced obesity model. These results showed that Nrf1 contributes to metabolic regulation, which gain-of-function develops diabetes mellitus in mice.


Assuntos
Glicemia/metabolismo , Fator 1 Relacionado a NF-E2/metabolismo , Animais , Peso Corporal , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Gorduras na Dieta/administração & dosagem , Resistência à Insulina , Fígado/metabolismo , Fator de Transcrição MafG/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 1 Relacionado a NF-E2/genética , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras/genética , Especificidade da Espécie , Transcrição Genética
17.
Toxicol Sci ; 141(1): 112-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24924401

RESUMO

The transcription factor Nrf2 (Nfe2l2 nuclear factor, erythroid 2-like 2) regulates gene expression directly, controlling pharmacological and toxicological responses. These processes may also be influenced by the structure of the hepatic vasculature, which distributes blood flow through compartmentalized microenvironments to maintain organismal stability. Castings of the hepatic portal vasculature of albino C57BL/6J but not ICR Nrf2(-/-) mice revealed a congenital intrahepatic shunt that was present in two thirds of Nrf2-disrupted mice. This shunt directly connected the portal vein to the inferior vena cava and displayed characteristics of a patent ductus venosus. Immunohistochemistry revealed that Nrf2(-/-) mice with an intrahepatic shunt manifest changes to hepatic oxygen and protein expression gradients when compared with wild-type (WT) and non-shunted Nrf2(-/-) mice. Centrilobular hypoxia found in WT and Nrf2(-/-) mice without shunts was reduced in Nrf2(-/-) livers with a shunt. Hepatic protein expression of phosphoenolpyruvate carboxykinase (Pepck), normally confined to the periportal zone, exhibited both periportal and centrilobular zonal expression in livers from Nrf2(-/-) mice with an intrahepatic shunt. Centrilobular expression of Cytochrome P450 2E1 (Cyp2e1) was diminished in shunted Nrf2(-/-) livers compared with WT and Nrf2(-/-) livers without shunts. The intrahepatic shunt in Nrf2(-/-) mice was further found to diminish acetaminophen hepatoxicity compared with WT and Nrf2(-/-) non-shunted mice following a 6 h challenge with 250 mg/kg acetaminophen. The presence of an intrahepatic shunt influences several physiological and pathophysiological properties of Nrf2(-/-) mice through changes in blood flow, hepatic oxygenation, and protein expression that extent beyond loss of canonical transactivation of Nrf2 target genes.


Assuntos
Malformações Arteriovenosas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocromo P-450 CYP2E1/genética , Modelos Animais de Doenças , Circulação Hepática , Fator 2 Relacionado a NF-E2/deficiência , Oxigênio/metabolismo , Acetaminofen/farmacocinética , Acetaminofen/toxicidade , Animais , Malformações Arteriovenosas/genética , Malformações Arteriovenosas/patologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Fígado/irrigação sanguínea , Fígado/enzimologia , Fígado/patologia , Circulação Hepática/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética
18.
Free Radic Biol Med ; 52(2): 444-51, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22107959

RESUMO

Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the cap'n'collar basic-region leucine-zipper transcription factor Nrf2 is a potential strategy to enhance expression of antioxidant and free radical detoxification gene products regulated by Nrf2. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer-chemopreventive agents. We describe the structure-activity relationships for a series of peptides based upon regions of the Nrf2 Neh2 domain, of varying length and sequence, that interact with the Keap1 Kelch domain and disrupt the interaction with Nrf2. We have also investigated sequestosome-1 (p62) and prothymosin-α sequences that have been reported to interact with Keap1. To achieve this we have developed a high-throughput fluorescence polarization (FP) assay to screen inhibitors. In addition to screening synthetic peptides, we have used a phage display library approach to identify putative peptide ligands with non-native sequence motifs. Candidate peptides from the phage display library screening protocol were evaluated in the FP assay to quantify their binding activity. Hybrid peptides based upon the Nrf2 "ETGE" motif and the sequestosome-1 "Keap1-interaction region" have superior binding activity compared to either native peptide alone.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Fator 2 Relacionado a NF-E2/química , Oligopeptídeos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Ligação Competitiva , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli , Polarização de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/biossíntese , Biblioteca de Peptídeos , Ligação Proteica , Precursores de Proteínas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteína Sequestossoma-1 , Timosina/análogos & derivados , Timosina/química
19.
PLoS One ; 6(10): e26884, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22046393

RESUMO

The Keap1-Nrf2 system serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than one hundred cytoprotective proteins, including antioxidants and phase 2 detoxifying enzymes. Since induction profiles of Nrf2 target genes have been studied exclusively in cultured cells, and not in animal models, their tissue-specificity has not been well characterized. In this paper, we examined and compared the tissue-specific expression of several Nrf2 target genes in zebrafish larvae by whole-mount in situ hybridization (WISH). Seven zebrafish genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) suitable for WISH analysis were selected from candidates for Nrf2 targets identified by microarray analysis. Tissue-restricted induction was observed in the nose, gill, and/or liver for all seven genes in response to Nrf2-activating compounds, diethylmaleate (DEM) and sulforaphane. The Nrf2 gene itself was dominantly expressed in these three tissues, implying that tissue-restricted induction of Nrf2 target genes is defined by tissue-specific expression of Nrf2. Interestingly, the induction of frrs1c and gclc in liver and nose, respectively, was quite low and that of hmox1a was restricted in the liver. These results indicate the existence of gene-specific variations in the tissue specificity, which can be controlled by factors other than Nrf2.


Assuntos
Citoproteção/genética , Regulação da Expressão Gênica/fisiologia , Fator 2 Relacionado a NF-E2/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Brânquias/metabolismo , Fígado/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Mucosa Nasal/metabolismo , Especificidade de Órgãos , Proteínas de Peixe-Zebra/agonistas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...