Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.192
Filtrar
1.
Life Sci ; 244: 117341, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31972208

RESUMO

Autophagy is a conserved "self-eating" recycling process which removes aggregated or misfolded proteins, or defective organelles, to maintain cellular hemostasis. In the autophagy-lysosome pathway (ALP), clearance of unwanted debris and materials occurs through the generation of the autophagosome, a complex of double-membrane bounded vesicles that form around cytosolic cargos and catabolize their contents by fusion to lysosomes. In tumors, autophagy has dichotomous functions via preventing tumor initiation but promoting tumor progression. The basic helix-loop-helix leucine zipper transcription factor EB (TFEB) activates the promoters of genes encoding for proteins, which participate in this cellular degradative system by regulating lysosomal biogenesis, lysosomal acidification, lysosomal exocytosis and autophagy. In humans, disturbances of ALP are related to various pathological conditions. Recently, TFEB dysregulation was found to have a crucial pathogenic role in different tumors by modulating tumor cell autophagy. Notably, in renal cell carcinomas, different TFEB gene fusions were reported to promote oncogenic features. In this review, we discuss the role of TFEB in human cancers with a special focus on potential diagnostic and therapeutic implications.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Humanos , Neoplasias/metabolismo
2.
Cell Physiol Biochem ; 54(1): 110-125, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31999897

RESUMO

BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells. METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned. RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin. CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Esfingolipídeos/metabolismo , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Colesterol/análise , Cromatografia Líquida de Alta Pressão , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Espectrometria de Massas , PPAR gama/genética , PPAR gama/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/análise , Esfingomielinas/análise
3.
Nat Commun ; 11(1): 479, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980602

RESUMO

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.


Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Escoliose/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Transporte Ativo do Núcleo Celular , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Mutação , Notocorda/anormalidades , Notocorda/metabolismo , Notocorda/ultraestrutura , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Escoliose/congênito , Escoliose/metabolismo , Transdução de Sinais , Coluna Vertebral/anormalidades , Coluna Vertebral/metabolismo , Fatores de Transcrição/metabolismo , Vacúolos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
4.
J Surg Res ; 246: 442-449, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31635836

RESUMO

BACKGROUND: MicroRNA (miRNA)-382-5p functions as an oncogenic miRNA in breast cancer. MXD1 was demonstrated to be one of its direct targets. However, the involvement of miRNA-382-5p/MXD1 axis in breast cancer remains unknown. The aim of this study was to investigate the expression pattern, clinical significance, and potential functions of miRNA-382-5p/MXD1 axis in breast cancer. MATERIALS AND METHODS: Quantitative polymerase chain reaction was performed to detect the expression levels of miRNA-382-5p and MXD1 messenger RNA (mRNA) in 96 pairs of breast cancer and matched noncancerous breast tissue samples from the same patients. Relationships between miRNA-382 expression, MXD1 expression, and combined miRNA-382-5p and MXD1 expression, and various clinicopathological characteristics of breast cancer were statistically evaluated, and their roles in breast cancer cell proliferation and invasion were also examined. RESULTS: Compared with noncancerous breast tissues, miRNA-382-5p expression was upregulated but MXD1 mRNA expression was downregulated in breast cancer tissues (both P < 0.01). High miRNA-382 expression, MXD1 expression, and combined miRNA-382-5p and low MXD1 expression were significantly associated with advanced tumor stage and the presence of lymph node metastasis (all P < 0.05). Overexpression of miRNA-382-5p dramatically reduced MXD1 mRNA and protein expression levels in breast cancer cells. miRNA-382-5p upregulation markedly enhanced breast cancer cell proliferation and invasion, while its downregulation inhibited these malignant phenotypes of breast cancer cells in vitro. Notably, overexpressed MXD1 reversed the effects of upregulated miRNA-382-5p on cell proliferation and invasion in vitro. CONCLUSIONS: The dysregulation of miRNA-382-5p-MXD1 axis may be involved in the development and aggressive progression of breast cancer. miRNA-382-5p may target MXD1, leading to cell invasion and proliferation in breast cancer cells in vitro, implying its potentials as a therapeutic target for this type of cancer.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Proliferação de Células/genética , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo
5.
Nat Commun ; 10(1): 5630, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822666

RESUMO

The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKß and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.


Assuntos
Autofagossomos/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Canais de Receptores Transientes de Potencial/metabolismo , Autofagossomos/ultraestrutura , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteína Beclina-1/metabolismo , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Mucolipidoses/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Fosfosserina/metabolismo , Canais de Receptores Transientes de Potencial/agonistas
6.
Int J Mol Sci ; 20(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623194

RESUMO

The worldwide increase in type 2 diabetes (T2D) is becoming a major health concern, thus searching for novel preventive and therapeutic strategies has become urgent. In last decade, the paralogous transcription factors MondoA and carbohydrate response element-binding protein (ChREBP) have been revealed to be central mediators of glucose sensing in multiple metabolic organs. Under normal nutrient conditions, MondoA/ChREBP plays vital roles in maintaining glucose homeostasis. However, under chronic nutrient overload, the dysregulation of MondoA/ChREBP contributes to metabolic disorders, such as insulin resistance (IR) and T2D. In this review, we aim to provide an overview of recent advances in the understanding of MondoA/ChREBP and its roles in T2D development. Specifically, we will briefly summarize the functional similarities and differences between MondoA and ChREBP. Then, we will update the roles of MondoA/ChREBP in four T2D-associated metabolic organs (i.e., the skeletal muscle, liver, adipose tissue, and pancreas) in physiological and pathological conditions. Finally, we will discuss the opportunities and challenges of MondoA/ChREBP as drug targets for anti-diabetes. By doing so, we highlight the potential use of therapies targeting MondoA/ChREBP to counteract T2D and its complications.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Transdução de Sinais , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Biomarcadores , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/etiologia , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Resistência à Insulina , Fígado/metabolismo , Pâncreas/metabolismo
7.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505737

RESUMO

Hyperglycemia is the major characteristic of diabetes mellitus, and a chronically high glucose (HG) level causes ß-cell glucolipotoxicity, which is characterized by lipid accumulation, impaired ß-cell function, and apoptosis. TXNIP (Thioredoxin-interacting protein) is a key mediator of diabetic ß-cell apoptosis and dysfunction in diabetes, and thus, its regulation represents a therapeutic target. Recent studies have reported that p90RSK is implicated in the pathogenesis of diabetic cardiomyopathy and nephropathy. In this study, we used FMK (a p90RSK inhibitor) to determine whether inhibition of p90RSK protects ß-cells from chronic HG-induced TXNIP expression and to investigate the molecular mechanisms underlying the effect of FMK on its expression. In INS-1 pancreatic ß-cells, HG-induced ß-cell dysfunction, apoptosis, and ROS generation were significantly diminished by FMK. In contrast BI-D1870 (another p90RSK inhibitor) did not attenuate HG-induced TXNIP promoter activity or TXNIP expression. In addition, HG-induced nuclear translocation of ChREBP and its transcriptional target molecules were found to be regulated by FMK. These results demonstrate that HG-induced pancreatic ß-cell dysfunction resulting in HG conditions is associated with TXNIP expression, and that FMK is responsible for HG-stimulated TXNIP gene expression by inactivating the regulation of ChREBP in pancreatic ß-cells. Taken together, these findings suggest FMK may protect against HG-induced ß-cell dysfunction and TXNIP expression by ChREBP regulation in pancreatic ß-cells, and that FMK is a potential therapeutic reagent for the drug development of diabetes and its complications.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ciclo Celular/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células Secretoras de Insulina/patologia , Ratos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
8.
Mol Cell ; 76(1): 110-125.e9, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31474573

RESUMO

Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.


Assuntos
Autofagia/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Senescência Celular/efeitos dos fármacos , Imunossenescência/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Espermidina/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Fatores Etários , Envelhecimento , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células HEK293 , Humanos , Memória Imunológica/efeitos dos fármacos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais
9.
Mol Cell Biol ; 39(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31501275

RESUMO

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Genes myc , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/farmacologia , Proteínas Recombinantes/farmacologia , Transcrição Genética , Ativação Transcricional
10.
Nat Commun ; 10(1): 3623, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399583

RESUMO

Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.


Assuntos
Autofagia/fisiologia , Epigênese Genética , Lisossomos/metabolismo , Biogênese de Organelas , Politetrafluoretileno/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Histona Desacetilases/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Células-Tronco , Transcrição Genética
11.
Nutrients ; 11(8)2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31405194

RESUMO

Ageing is a major risk factor for the development of metabolic disorders linked to dyslipidemia, usually accompanied by increased adiposity. The goal of this work was to investigate whether avoiding an excessive increase in adiposity with ageing, via moderate chronic food restriction (FR), ameliorates postprandial dyslipidemia in a rat model of metabolic syndrome associated with ageing. Accordingly, we performed an oral lipid loading test (OLLT) in mature middle-aged (7 months) and middle-old-aged (24 months) Wistar rats fed ad libitum (AL) or under moderate FR for 3 months. Briefly, overnight fasted rats were orally administered a bolus of extra-virgin olive oil (1 mL/Kg of body weight) and blood samples were taken from the tail vein before fat load (t = 0) and 30, 60, 90, 120, 180, and 240 min after fat administration. Changes in serum lipids, glucose, insulin, and glucagon levels were measured at different time-points. Expression of liver and adipose tissue metabolic genes were also determined before (t = 0) and after the fat load (t = 240 min). Postprandial dyslipidemia progressively increased with ageing and this could be associated with hepatic ChREBP activity. Interestingly, moderate chronic FR reduced adiposity and avoided excessive postprandial hypertriglyceridemia in 7- and 24-month-old Wistar rats, strengthening the association between postprandial triglyceride levels and adiposity. The 24-month-old rats needed more insulin to maintain postprandial normoglycemia; nevertheless, hyperglycemia occurred at 240 min after fat administration. FR did not alter the fasted serum glucose levels but it markedly decreased glucagon excursion during the OLLT and the postprandial rise of glycemia in the 24-month-old rats, and FGF21 in the 7-month-old Wistar rats. Hence, our results pointed to an important role of FR in postprandial energy metabolism and insulin resistance in ageing. Lastly, our data support the idea that the vWAT might function as an ectopic site for fat deposition in 7-month-old and in 24-month-old Wistar rats that could increase their browning capacity in response to an acute fat load.


Assuntos
Envelhecimento/metabolismo , Dieta com Restrição de Gorduras/métodos , Dislipidemias/etiologia , Síndrome Metabólica/metabolismo , Período Pós-Prandial , Adiposidade , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glicemia/análise , Gorduras na Dieta/metabolismo , Modelos Animais de Doenças , Glucagon/sangue , Insulina/sangue , Lipídeos/sangue , Fígado/metabolismo , Síndrome Metabólica/etiologia , Ratos , Ratos Wistar , Triglicerídeos/sangue
12.
Biochem Soc Trans ; 47(4): 1173-1185, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31383818

RESUMO

Lysosomes are the main degradative compartments of mammalian cells and serve as platforms for cellular nutrient signaling and sterol transport. The diverse functions of lysosomes and their adaptation to extracellular and intracellular cues are tightly linked to the spatiotemporally controlled synthesis, turnover and interconversion of lysosomal phosphoinositides, minor phospholipids that define membrane identity and couple membrane dynamics to cell signaling. How precisely lysosomal phosphoinositides act and which effector proteins within the lysosome membrane or at the lysosomal surface recognize them is only now beginning to emerge. Importantly, mutations in phosphoinositide metabolizing enzyme cause lysosomal dysfunction and are associated with numerous diseases ranging from neurodegeneration to cancer. Here, we discuss the phosphoinositides and phosphoinositide metabolizing enzymes implicated in lysosome function and homeostasis and outline perspectives for future research.


Assuntos
Homeostase , Lisossomos/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Autofagossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Transporte Biológico , Humanos , Membranas Intracelulares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Esteróis/metabolismo
13.
J Exp Clin Cancer Res ; 38(1): 340, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387632

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma is one of the most aggressive cancers, with a 5-year survival rate of less than 8%. The complicated tumor microenvironment, particularly TGF-ß, provides possible convenience for the progression of PC cells. TGF-ß regulates critical cellular processes, including autophagy. However, the mechanism and effects of TGF-ß-mediated autophagy are still poorly understood. METHODS: Bioinformatics analysis, western blot, transmission electron microscopy and confocal microscopy were used to identify that TFEB is the key factors in TGF-ß-induced autophagy. The biological effects of TFEB-driven autophagy were investigated in vitro using transwell and wound healing assays and in vivo using liver metastasis and LSL-KrasG12D/Pdx1-Cre mice models. Luciferase assays and motif analysis were used to assess regulation of RAB5A gene promoter activity by TGF-ß-induced TFEB. TFEB levels were measured by real-time PCR, western blot and immunohistochemical staining in clinical pancreatic ductal adenocarcinoma tissues. RESULTS: We demonstrated that TGF-ß induces TFEB expression via the canonical smad pathway in Smad4-positive PC cells and facilitates TFEB-mediated autophagic activation. TFEB-driven autophagy caused by TGF-ß regulates RAB5A-dependent endocytosis of Itgα5 and promotes progression of PC cells. We further showed that enhanced TFEB expression and its direct target RAB5A both predict poor prognosis in PC patients. CONCLUSIONS: Our findings reveal TFEB-driven autophagy is required for TGF-ß induced migration and metastasis of PC cells by promoting endocytosis of Itgα5ß1 and focal adhesion disassembly through the TGF-ß-TFEB-RAB5A axis. Our results highlight the potential utility of suppressing TFEB-driven autophagy to block PC metastasis.


Assuntos
Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Endocitose/genética , Feminino , Expressão Gênica , Humanos , Integrinas/genética , Integrinas/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/patologia , Interferência de RNA , Proteínas Smad/metabolismo
14.
BMC Genomics ; 20(1): 673, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31455224

RESUMO

BACKGROUND: Herbivorous insects can have a profound impact on plant growth performance. In some years, canopy damage in poplar plantations exceeds 50% of the total leaf surface, thereby possibly compromising carbon fixation and biomass yield. To assess the transcriptional response of elite poplar clones to insect feeding and to test whether this response varies between different genotypes, we performed an RNA-sequencing experiment. We deeply sequenced the transcriptomes of eight elite clones belonging to three poplar species (Populus trichocarpa, P. nigra and P. maximowiczii), under Phratora vitellinae feeding and control conditions. This allowed us to precisely quantify transcript levels of about 24,000 expressed genes. RESULTS: Our data reveal a striking overall up-regulation of gene expression under insect attack in all eight poplar clones studied. The up-regulated genes were markedly enriched for the biological process 'regulation of transcription' indicating a highly concerted restructuring of the transcriptome. A search for potential cis-regulatory elements (CREs) that may be involved in this process identified the G-box (CACGTG) as the most significant motif in the promoters of the induced genes. In line with the role of the G-box in jasmonate (JA)-mediated activation of gene expression by MYC2, several genes involved in JA biosynthesis and signaling were up-regulated in our dataset. A co-expression network analysis additionally highlighted WRKY transcription factors. Within the most prominent expression module, WRKYs were strongly overrepresented and occupied several network hubs. Finally, the insect-induced genes comprised several protein families known to be involved in plant defenses, e.g. cytochrome P450s, chitinases and protease inhibitors. CONCLUSIONS: Our data represent a comprehensive characterization of the transcriptional response of selected elite poplar clones to insect herbivory. Our results suggest that the concerted up-regulation of gene expression is controlled by JA signaling and WRKY transcription factors, and activates several defense mechanisms. Our data highlight potential targets of selection and may thus contribute to breeding insect-resistant poplar clones.


Assuntos
Regulação da Expressão Gênica de Plantas , Herbivoria , Populus/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Quitinases/metabolismo , Ciclopentanos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Insetos/fisiologia , Oxilipinas/metabolismo , Populus/metabolismo , Inibidores de Proteases/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Transcriptoma , Regulação para Cima
16.
Food Chem Toxicol ; 131: 110591, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31212009

RESUMO

Kidney ischemia reperfusion injury (IRI) is an acute kidney injury associated with high number of mortality. We have examined the molecular mechanism and found that oxidative stress and hypoxia leads to induction of autophagy. In IRI induced autophagy, TFEB translocated to nucleus in response to IRI and induced a number of target genes of Coordinated Lysosomal Expression and Regulation (CLEAR) network. Real-time PCR analyses result showed IRI dependent increase in mRNA level to lysosomal hydrolases (Ctsa, Psap), lysosomal membranes (Lamp1), lysosomal acidification (Atp6ap1) non-lysosomal proteins involved in lysosomal biogenesis (M6pr, Nagpa) and autophagy (Becn1, VPS11). Overall, both lysosomal biogenesis and autophagy pathways were induced. Two key players of TFEB dependent proteins in autophagy, LAMP1 and BECN1 were verified by protein analyses. Pretreatment with urolithin A promoted autophagy and attenuated renal injury in kidney IRI and thus inverse relationship existed between TFEB-CLEAR pathway and kidney injury. Urolithin A also attenuated IRI induced pro-inflammatory cytokines TNFα, IL1ß, MIP1α and MIP2 mRNA and associated kidney injury. Overall, our results explored the understanding of autophagy and CLEAR network to kidney IRI and those insights may help to develop new therapeutic strategies to protect against IRI.


Assuntos
Lesão Renal Aguda/prevenção & controle , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cumarínicos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Lesão Renal Aguda/fisiopatologia , Animais , Autofagia/fisiologia , Núcleo Celular/metabolismo , Citocinas/metabolismo , Inflamação/prevenção & controle , Rim/patologia , Rim/fisiopatologia , Lisossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Traumatismo por Reperfusão/fisiopatologia
17.
Nat Plants ; 5(6): 616-625, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182849

RESUMO

The lipid-derived hormone jasmonate (JA) regulates plant immunity and adaptive growth by triggering a genome-wide transcriptional programme. In Arabidopsis thaliana, JA-triggered transcriptional programming is largely orchestrated by the master transcription factor MYC2. The function of MYC2 is dependent on its physical interaction with the MED25 subunit of the Mediator transcriptional co-activator complex. Here we report the identification of JA enhancers (JAEs) through profiling the occupancy pattern of MYC2 and MED25. JA regulates the dynamic chromatin looping between JAEs and their promoters in a MED25-dependent manner, while MYC2 auto-regulates itself through JAEs. Interestingly, the JAE of the MYC2 locus (named ME2) positively regulates MYC2 expression during short-term JA responses but negatively regulates it during constant JA responses. We demonstrate that new gene editing tools open up new avenues to elucidate the in vivo function of enhancers. Our work provides a paradigm for functional study of plant enhancers in the regulation of specific physiological processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclopentanos/metabolismo , Proteínas Nucleares/metabolismo , Oxilipinas/metabolismo , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Arseniato Redutases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
18.
Cell Mol Biol Lett ; 24: 33, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31160892

RESUMO

Background: Induction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB. Methods: Expression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes. Results: Our results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes. Conclusions: Collectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Epitélio Pigmentado da Retina/patologia , Estresse Fisiológico , Adulto , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estresse Fisiológico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
19.
Int J Mol Med ; 44(2): 447-456, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173156

RESUMO

The aim of the present study was to investigate the effects of transcription factor EB (TFEB) overexpression on oxidative stress, mitochondrial function and apoptosis in podocytes induced with high glucose. High glucose­induced time­dependent changes in TFEB expression were identified and nuclear translocation of TFEB was observed in podocytes. Overexpression of TFEB markedly reduced high glucose­induced oxidative stress in podocytes, and increased the expression of superoxide dismutase 2 and heme oxygenase 1 antioxidant enzymes. It was further observed that TFEB overexpression could partially restore the expression of peroxisome proliferator­activated receptor­Î³ coactivator­1α, transcription factor A, mitochondrial, and cytochrome c oxidase subunit 4, thereby enhancing mitochondrial biosynthesis. Furthermore, overexpression of TFEB reduced mitochondrial swelling and fragmentation, restored mitochondrial membrane potential, and contributed to the restoration of mitochondrial function. By overexpressing TFEB, it was revealed that TFEB increased the ratios of phosphorylated (p)­Akt/Akt and p­Bad/Bad, and the expression of downstream Bcl­xl, and reduced the ratio of Bax/Bcl­2 and the expression of cleaved­caspase­3 compared with high glucose­treatment. Furthermore, when the Akt phosphorylation inhibitor Ly294002 was added, the improvement by TFEB to high glucose­induced apoptosis was significantly reduced. These findings suggest that overexpressing TFEB could reduce the production of reactive oxygen species in podocytes in a high glucose environment, relieve oxidative stress, promote mitochondrial biogenesis and renewal functions, and reduce high glucose­induced podocyte apoptosis by activating the Akt/Bad pathway.


Assuntos
Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glucose/metabolismo , Estresse Oxidativo , Podócitos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Podócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
20.
J Exp Clin Cancer Res ; 38(1): 239, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174569

RESUMO

BACKGROUND: Temozolomide-perillyl alcohol conjugate (NEO212), a novel temozolomide (TMZ) analog, was previously reported to exert its anti-cancer effect in non-small cell lung cancer (NSCLC), and human nasopharyngeal carcinoma (NPC), etc.. In the current study, we intend to illuminate the potential anticancer property and the underly mechanisms of NEO212 in ovarian cancer cells. METHODS: The cytotoxicity of NEO212 was detected by MTT, colony formation analysis and xenograft model. The proteins involved in cell proliferation, DNA damage, autophagy and lysosomal function were detected by western blots; mitochondria, lysosome and autophagosome were visualized by TEM and/or immunofluorescence; Apoptosis, cell cycle analysis and mitochondrial transmembrane potential were detected by flow cytometry. TFEB translocation was detected by immunofluorescence and western blot. RESULTS: NEO212 has the potential anticancer property in ovarian cancer cells, as evidence from cell proliferation inhibition, G2/M arrest, DNA damage, xenograft, mitochondrial dysfunction and apoptosis. Importantly, we observed that although it induced significant accumulation of autophagosomes, NEO212 quenched GFP-LC3 degradation, down-regulated a series of lysosome related gene expression and blocked the autophagic flux, which significantly facilitated it induced apoptosis and was largely because it inhibited the nuclear translocation of transcription factor EB (EB). CONCLUSIONS: NEO212 inhibited TFEB translocation, and impaired the lysosomal function, implying NEO212 might avoid from autophagy mediated chemo-resistance, thus proposing NEO212 as a potential therapeutic candidate for ovarian cancer.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Dacarbazina/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Antineoplásicos/uso terapêutico , Autofagossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Dano ao DNA/efeitos dos fármacos , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/ultraestrutura , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transporte Proteico , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA