RESUMEN

Alcohol is a well-known risk factor for hepatocellular carcinoma. Autophagy plays a dual role in liver cancer, as it suppresses tumor initiation and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy, which is impaired in alcohol-related liver disease. However, the role of TFEB in alcohol-associated liver carcinogenesis is unknown. Liver-specific Tfeb knockout (KO) mice and their matched wild-type (WT) littermates were injected with the carcinogen diethylnitrosamine (DEN), followed by chronic ethanol feeding. The numbers of both total and larger tumors increased significantly in DEN-treated mice fed ethanol diet than in mice fed control diet. Although the number of tumors was not different between WT and L-Tfeb KO mice fed either control or ethanol diet, the number of larger tumors was less in L-Tfeb KO mice than in WT mice. No differences were observed in liver injury, steatosis, inflammation, ductular reaction, fibrosis, and tumor cell proliferation in DEN-treated mice fed ethanol. However, the levels of glypican 3, a marker of malignant hepatocellular carcinoma, markedly decreased in DEN-treated L-Tfeb KO mice fed ethanol in comparison to the WT mice. These findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which is attenuated by genetic deletion of hepatic TFEB.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Carcinogénesis/metabolismo , Carcinogénesis/patología , Etanol/efectos adversos , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Consumo de Bebidas Alcohólicas/efectos adversos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular , Dieta Occidental , Dietilnitrosamina , Eliminación de Gen , Inflamación/patología , Hígado/patología , Hígado/ultraestructura , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Carga Tumoral

RESUMEN

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.

Asunto(s)

ADN/química , Lisosomas/metabolismo , Nanopartículas/química , Neoplasias/patología , Macrófagos Asociados a Tumores/metabolismo , Animales , Antígenos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Terapia Combinada , Reactividad Cruzada/inmunología , Ciclofosfamida , Femenino , Humanos , Inmunidad , Ratones Endogámicos C57BL , Neoplasias/inmunología , Proteómica

RESUMEN

AIMS: In the present study, we investigated the effect of carbohydrate responsive element binding protein (ChREBP) on the TXNIP/oxidative stress and apoptosis in diabetic nephropathy. METHODS: ChREBP-/- mice (8-week old) were produced using the CRISPR/Cas9 gene editing approach. Diabetes was induced in C57BL/6 mice with streptozotocin. HK-2 cells was transfected with plasmid containing either ChREBP shRNA or TXNIP siRNA. RESULTS: Renal expression of ChREBP and thioredoxin-interacting protein (TXNIP) was increased in patients with type 2 diabetes mellitus (T2DM) and diabetic mice. ChREBP deficiency improved renal function, apoptosis as well as endoplasmic reticulum (ER) stress in diabetic mice. In addition, ChREBP deficiency prevented expression levels of TXNIP and NADPH oxidase 4 (Nox4), 8-hydroxydeoxyguanosine (8-OHdG) and heme oxygenase-1 (HO-1) in diabetic kidneys. The increased urinary 8-OHdG level induced by diabetes was also attenuated in ChREBP deficiency mice. Similarly, HG was shown to induce ChREBP expression and nuclear translocation in HK-2 cells. HG-induced apoptosis was inhibited by transfection of ChREBP shRNA plasmid. Moreover, we found that knockdown of ChREBP suppressed HG-induced TXNIP and Nox4 expression, reactive oxygen species (ROS) generation and ER stress in HK-2 cells. Furthermore, TXNIP knockdown effectively abrogated HG-induced apoptosis in HK-2 cells. CONCLUSIONS: These results suggest that ChREBP deficiency prevents diabetes-induced apoptosis via inhibiting oxidative stress and ER stress, highlighting ChREBP as a potential therapy target for diabetic nephropathy.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas Portadoras , Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Tiorredoxinas , Animales , Apoptosis/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Glucemia/análisis , Proteínas Portadoras/biosíntesis , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/fisiología , Edición Génica , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Riñón/citología , Riñón/metabolismo , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Tiorredoxinas/biosíntesis

RESUMEN

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that increases the transcription of multiple genes. ChREBP is highly localized in the liver, where it upregulates the expression of genes that code for glycolytic and lipogenic enzymes, resulting in the conversion of excess carbohydrate into storage fat. ChREBP knockout (KO) mice display an anti-obese phenotype. However, at this time, role of ChREBP in adipose tissue remains unclear. Therefore, the energy metabolism and morphology of mitochondrial brown adipose tissue (BAT) in ChREBP KO mice was examined. We found increased expression levels of electron transport system proteins including the mitochondrial uncoupling protein (UCP1), and mitochondrial structural alterations such as dysplasia of the cristae and the presence of small mitochondria in BAT of ChREBP KO mice. Mass spectrometry analyses revealed that fatty acid synthase was absent in the BAT of ChREBP KO mice, which probably led to a reduction in fatty acids and cardiolipin, a regulator of various mitochondrial events. Our study clarified the new role of ChREBP in adipose tissue and its involvement in mitochondrial function. A clearer understanding of ChREBP in mitochondria could pave the way for improvements in obesity management.

Asunto(s)

Tejido Adiposo Pardo/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Metabolismo Energético , Mitocondrias/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Obesidad/genética , Obesidad/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo

RESUMEN

Carbohydrate response element-binding protein (ChREBP) is critical in the regulation of fatty acid and triglyceride synthesis in the liver. Interestingly, Chrebp-/- mice show reduced levels of plasma cholesterol, which is critical for steroid hormone synthesis in adrenal glands. Furthermore, Chrebp mRNA expression was previously reported in human adrenal glands. Thus, it remains to be investigated whether ChREBP plays a role directly or indirectly in steroid hormone synthesis and release in adrenal glands. In the present study, we find that Chrebp mRNA is expressed in mouse adrenal glands and that ChREBP binds to carbohydrate response elements. Histological analysis of Chrebp-/- mice shows no adrenal hyperplasia and less oil red O staining compared with that in WT mice. In adrenal glands of Chrebp-/- mice, expression of Fasn and Scd1, two enzymes critical for fatty acid synthesis, was substantially lower and triglyceride content was reduced. Expression of Srebf2, a key transcription factor controlling synthesis and uptake of cholesterol and the target genes, was upregulated, while cholesterol content was not significantly altered in the adrenal glands of Chrebp-/- mice. Adrenal corticosterone content and plasma adrenocorticotropic hormone and corticosterone levels were not significantly altered in Chrebp-/- mice. Consistently, expression of genes related to steroid hormone synthesis was not altered. Corticosterone secretion in response to two different stimuli, namely 24-h starvation and cosyntropin administration, was also not altered in Chrebp-/- mice. Taking these results together, corticosterone synthesis and release were not affected in Chrebp-/- mice despite reduced plasma cholesterol levels.

Asunto(s)

Glándulas Suprarrenales/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Colesterol/sangre , Corticosterona/biosíntesis , Lipogénesis , Animales , Masculino , Ratones Endogámicos C57BL

RESUMEN

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

Asunto(s)

Síndrome de Birt-Hogg-Dubé/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patología , Línea Celular , Modelos Animales de Enfermedad , Activación Enzimática , Células HeLa , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Especificidad por Sustrato , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética

RESUMEN

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. METHODS: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. RESULTS: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-ß-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-ß-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. CONCLUSIONS: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-ß-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Asunto(s)

2-Hidroxipropil-beta-Ciclodextrina/uso terapéutico , Aneurisma de la Aorta Abdominal/prevención & control , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Modelos Animales de Enfermedad , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , 2-Hidroxipropil-beta-Ciclodextrina/farmacología , Aminopropionitrilo/toxicidad , Aneurisma Roto/etiología , Angiotensina II/toxicidad , Animales , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Apoptosis/efectos de los fármacos , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Colesterol/metabolismo , Regulación hacia Abajo , Evaluación Preclínica de Medicamentos , Mutación con Ganancia de Función , Regulación de la Expresión Génica , Vectores Genéticos/toxicidad , Humanos , Mutación con Pérdida de Función , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Transcriptoma/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Autofagia/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Senescencia Celular/efectos de los fármacos , Inmunosenescencia/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Espermidina/farmacología , Inmunidad Adaptativa/efectos de los fármacos , Factores de Edad , Envejecimiento , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células HEK293 , Humanos , Memoria Inmunológica/efectos de los fármacos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. unc13d-null cells showed impaired endosomal trafficking and defective endocytic flux. The defective phenotypes were rescued by the expression of UNC13D but not by its STX7-binding-deficient mutant. This defective endosomal function in UNC13D-deficient cells resulted in increased autophagic flux, increased long-lived protein degradation, decreased SQSTM1/p62 protein levels and increased autolysosome formation as determined by biochemical, microscopy and structural methods. The autophagic phenotype was not associated with increased recruitment of the UNC13D-binding proteins and autophagy regulators, RAB11 or VAMP8, but was caused, at least in part, by TFEB-mediated upregulation of a subset of autophagic and lysosomal genes, including Atg9b. Downregulation of TFEB decreased Atg9b levels and decreased macroautophagy in unc13d-null cells. UNC13D upregulation corrected the defects in endolysosomal trafficking and decreased the number of accumulated autophagosomes in a cellular model of the lysosomal-storage disorder cystinosis, under both fed and starvation conditions, identifying UNC13D as an important new regulatory molecule of autophagy regulation in cells with lysosomal disorders. Abbreviations ACTB: actin, beta; CTSB: cathepsin B; EEA1: early endosome antigen 1; ESCRT: endosomal sorting complex required for transport; FHL3: familial hemophagocytic; lymphohistiocytosis type 3; HEX: hexosaminidase; HLH: hemophagocytic lymphohistiocytosis; LSD: lysosomal storage disorder; MEF: mouse embryonic fibroblast; SEM: standard errors of the mean; SNARE: soluble n-ethylmaleimide-sensitive-factor attachment receptor; STX: syntaxin; SYT7: synaptotagmin VII; TFE3: transcription factor E3; TFEB: transcription factor EB; TIRF: total internal reflection fluorescence ULK1: unc-51 like kinase 1; UNC13D: unc-13 homolog d; VAMP: vesicle-associate membrane protein; WT: wild-type.

Asunto(s)

Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Animales , Autofagosomas/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Transporte Biológico/genética , Células Cultivadas , Cistinosis/genética , Cistinosis/metabolismo , Cistinosis/patología , Endosomas/genética , Células HEK293 , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/genética , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética

RESUMEN

Oncogenic Ras upregulates aerobic glycolysis to meet the bioenergetic and biosynthetic demands of rapidly growing cells. In contrast, thioredoxin-interacting protein (TXNIP) is a potent inhibitor of glucose uptake and is frequently downregulated in human cancers. Our laboratory previously discovered that Ras activation suppresses TXNIP transcription and translation. In this study, we developed a system to study how Ras affects TXNIP translation in the absence of transcriptional effects. We show that whereas Ras drives a global increase in protein translation, it suppresses TXNIP protein synthesis by reducing the rate at which ribosomes transit the coding region of TXNIP mRNA. To investigate the underlying mechanism(s), we randomized or optimized the codons in the TXNIP message without altering the TXNIP primary amino acid sequence. Translation from these mRNA variants was still repressed by Ras, implying that mRNA secondary structure, microRNAs (miRNAs), RNA binding proteins, or codon usage does not contribute to the blockade of TXNIP synthesis. Rather, we show that the N terminus of the growing TXNIP polypeptide is the target for Ras-dependent translational repression. Our work demonstrates how Ras suppresses TXNIP translation elongation in the face of a global upregulation of protein synthesis and provides new insight into Ras-dependent metabolic reprogramming.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células Cultivadas , Regulación hacia Abajo , Técnicas de Inactivación de Genes , Genes ras , Humanos , Ratones , Extensión de la Cadena Peptídica de Translación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribosomas/genética , Ribosomas/metabolismo

RESUMEN

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/patología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Proteínas de Unión al ARN

RESUMEN

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn's disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Mucosa Intestinal/patología , Animales , Apolipoproteína A-I/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Femenino , Regulación de la Expresión Génica , Homeostasis , Mucosa Intestinal/metabolismo , Masculino , Ratones , Células de Paneth/metabolismo

RESUMEN

The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence.

Asunto(s)

Adaptación Fisiológica , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Fúngicas/metabolismo , Histoplasma/metabolismo , Histoplasma/patogenicidad , Oxígeno/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Ergosterol/biosíntesis , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Histoplasma/efectos de los fármacos , Histoplasma/genética , Itraconazol/farmacología , Macrófagos/citología , Macrófagos/microbiología , Ratones , Virulencia

RESUMEN

Aggregation of α-synuclein (α-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson's disease (PD). The formation of α-syn aggregates results from aberrant accumulation of misfolded α-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of α-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated α-syn by inducing autophagic clearance of α-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-ß-cyclodextrin promotes autophagic clearance of aggregated α-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of α-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of α-syn aggregates.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Agregado de Proteínas/efectos de los fármacos , Agregado de Proteínas/genética , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina , Autofagia/efectos de los fármacos , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Línea Celular Tumoral , Silenciador del Gen , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , ARN Interferente Pequeño/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , beta-Ciclodextrinas/farmacología

RESUMEN

BACKGROUND: Cerebral palsy (CP) is a heterogeneous neurodevelopmental disorder associated with intellectual disability in one-third of cases. Recent findings support Mendelian inheritance in subgroups of patients with the disease. The purpose of this study was to identify a novel genetic cause of paraplegic CP with intellectual disability in a consanguineous Pakistani family. METHODS: We performed whole-exome sequencing (WES) in two brothers with CP and intellectual disability. Analysis of AP4M1 mRNA was performed using quantitative real-time PCR on total RNA from cultured fibroblasts. The brothers were investigated clinically and by MRI. RESULTS: We identified a novel homozygous AP4M1 mutation c.194_195delAT, p.Y65Ffs*50 in the affected brothers. Quantitative RT-PCR analysis showed markedly reduced AP4M1 mRNA levels suggesting partial non-sense mediated mRNA decay. Several clinical and MRI features were consistent with AP-4 complex deficiency. However, in contrast to previously reported cases with AP4M1 mutations our patients show an aggressive behavior and a relatively late onset of disease. CONCLUSION: This study shows an AP4M1 mutation associated with aggressive behavior in addition to mild dysmorphic features, intellectual disability, spastic paraparesis and reduced head circumference. Our findings expand the clinical spectrum associated with AP-4 complex deficiency and the study illustrates the importance of MRI and WES in the diagnosis of patients with CP and intellectual disability.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Parálisis Cerebral/genética , Mutación , Adolescente , Parálisis Cerebral/complicaciones , Niño , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Proteínas de Unión al ADN , Genes Recesivos , Homocigoto , Humanos , Lactante , Discapacidad Intelectual/etiología , Discapacidad Intelectual/genética , Imagen por Resonancia Magnética , Masculino , Linaje , ARN Mensajero/metabolismo , Proteínas de Unión al ARN

RESUMEN

MondoA is a basic helix-loop-helix (bHLH)/leucine zipper (ZIP) transcription factor that is expressed predominantly in skeletal muscle. Studies in vitro suggest that the Max-like protein X (MondoA:Mlx) heterodimer senses the intracellular energy status and directly targets the promoter region of thioredoxin interacting protein (Txnip) and possibly glycolytic enzymes. We generated MondoA-inactivated (MondoA-/-) mice by gene targeting. MondoA-/- mice had normal body weight at birth, exhibited normal growth and appeared to be healthy. However, they exhibited unique metabolic characteristics. MondoA-/- mice built up serum lactate and alanine levels and utilized fatty acids for fuel during exercise. Gene expression and promoter analysis suggested that MondoA functionally represses peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α)-mediated activation of pyruvate dehydrogenase kinase 4 (PDK-4) transcription. PDK4 normally down-regulates the activity of pyruvate dehydrogenase, an enzyme complex that catalyses the decarboxylation of pyruvate to acetyl-CoA for entry into the Krebs cycle; in the absence of MondoA, pyruvate is diverted towards lactate and alanine, both products of glycolysis. Dynamic testing revealed that MondoA-/- mice excel in sprinting as their skeletal muscles display an enhanced glycolytic capacity. Our studies uncover a hitherto unappreciated function of MondoA in fuel selection in vivo. Lack of MondoA results in enhanced exercise capacity with sprinting.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Resistencia Física/fisiología , Animales , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Condicionamiento Físico Animal/métodos

RESUMEN

Embryonic stem cells (ESCs) can self-renew indefinitely under the governance of ESC-specific transcriptional circuitries in which each transcriptional factor regulates distinct or overlapping sets of genes with other factors. c-Myc is a key player that is crucially involved in maintaining the undifferentiated state and the self-renewal of ESCs. However, the mechanism by which c-Myc helps preserve the ESC status is still poorly understood. Here we addressed this question by performing loss-of-function studies with the Max gene, which encodes the best-characterized partner protein for all Myc family proteins. Although Myc/Max complexes are widely regarded as crucial regulators of the ESC status, our data revealed that ESCs do not absolutely require these complexes in certain contexts and that this requirement is restricted to empirical ESC culture conditions without a MAPK inhibitor.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Animales , Apoptosis/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Diferenciación Celular/genética , Proliferación Celular , Supervivencia Celular , Activación Enzimática , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Modelos Biológicos

RESUMEN

Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4(GFPCre) mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Tejido Conectivo/fisiología , Desarrollo de Músculos/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Femenino , Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Desarrollo de Músculos/genética , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Embarazo , Transducción de Señal , Factor de Transcripción 4 , beta Catenina/metabolismo

RESUMEN

During brain development, neurons and glia are generated from a germinal zone containing both neural stem cells (NSCs) and more limited intermediate neural progenitors (INPs). The signalling events that distinguish between these two proliferative neural cell types remain poorly understood. The Notch signalling pathway is known to maintain NSC character and to inhibit neurogenesis, although little is known about the role of Notch signalling in INPs. Here we show that both NSCs and INPs respond to Notch receptor activation, but that NSCs signal through the canonical Notch effector C-promoter binding factor 1 (CBF1), whereas INPs have attenuated CBF1 signalling. Furthermore, whereas knockdown of CBF1 promotes the conversion of NSCs to INPs, activation of CBF1 is insufficient to convert INPs back to NSCs. Using both transgenic and transient in vivo reporter assays we show that NSCs and INPs coexist in the telencephalic ventricular zone and that they can be prospectively separated on the basis of CBF1 activity. Furthermore, using in vivo transplantation we show that whereas NSCs generate neurons, astrocytes and oligodendrocytes at similar frequencies, INPs are predominantly neurogenic. Together with previous work on haematopoietic stem cells, this study suggests that the use or blockade of the CBF1 cascade downstream of Notch is a general feature distinguishing stem cells from more limited progenitors in a variety of tissues.

Asunto(s)

Neuronas/citología , Neuronas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Células Cultivadas , Proteínas Fluorescentes Verdes/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Ratones , Telencéfalo/metabolismo

RESUMEN

Myc is the most frequently deregulated oncogene in human tumors. The protein belongs to the Myc/Max/Mxd network of transcriptional regulators important for cell growth, proliferation, differentiation, and apoptosis. The ratio between Mnt/Max and c-Myc/Max on the 5'-CACGTG-3' E-box sequence at shared target genes is of great importance for cell cycle progression and arrest. Serum stimulation of quiescent cells results in phosphorylation of Mnt and disruption of the critical Mnt-mSin3-HDAC1 interaction. This in turn leads to increased expression of the Myc/Mnt target gene cyclin D2. It is therefore possible that Myc function relies on its ability to overcome transcriptional repression by Mnt and that relief of Mnt-mediated transcriptional repression is of greater importance for regulation of target genes than the sole activation by Myc. In addition, Mnt has many features of a tumor suppressor and may thus be nonfunctional or inactivated in human tumors. In summary, accumulating evidence supports the model of Mnt as the key regulator of the network in vivo.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Represoras/fisiología , Animales , Antineoplásicos/farmacología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/antagonistas & inhibidores , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , División Celular , Sistemas de Liberación de Medicamentos , Redes Reguladoras de Genes , Genes Supresores de Tumor , Genes myc , Humanos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Transcripción Genética