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1.
Cell Physiol Biochem ; 52(6): 1569-1583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31145841

RESUMO

BACKGROUND/AIMS: Shear stress plays major roles in developmental angiogenesis, particularly in blood vessel remodeling and maturation but little is known about the shear stress sensors involved in this process. Our recent study identified endothelial Kir2.1 channels as major contributors to flow-induced vasodilation, a hallmark of the endothelial flow response. The goal of this study is to establish the role of Kir2.1 in the regulation of retinal angiogenesis. METHODS: The retina of newly born Kir2.1+/- mice were used to investigate the sprouting angiogenesis and remodeling of newly formed branched vessels. The structure, blood density and mural cell coverage have been evaluated by immunohistochemistry of the whole-mount retina. Endothelial cell alignment was assessed using CD31 staining. The experiments with flow-induced vasodilation were used to study the cerebrovascular response to flow. RESULTS: Using Kir2.1-deficient mice, we show that the retinas of Kir2.1+/- mice have higher vessel density, increased lengths and increased number of the branching points, as compared to WT littermates. In contrast, the coverage by αSMA is decreased in Kir2.1+/- mice while pericyte coverage does not change. Furthermore, to determine whether deficiency of Kir2.1 affects vessel pruning, we discriminated between intact and degraded vessels or "empty matrix sleeves" and found a significant reduction in the number of empty sleeves on the peripheral part of the retina or "angiogenic front" in Kir2.1+/- mice. We also show that Kir2.1 deficiency results in decreased endothelial alignment in retinal endothelium and impaired flow-induced vasodilation of cerebral arteries, verifying the involvement of Kir2.1 in shear-stress sensing in retina and cerebral circulation. CONCLUSION: This study shows that shear-stress sensitive Kir2.1 channels play an important role in pruning of excess vessels and vascular remodeling during retinal angiogenesis. We propose that Kir2.1 mediates the effect of shear stress on vessel maturation.


Assuntos
Neovascularização Patológica/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Estresse Mecânico , Animais , Artérias Cerebrais/metabolismo , Artérias Cerebrais/patologia , Células Endoteliais/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Retina/metabolismo , Retina/patologia
2.
Science ; 364(6437): 283-285, 2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31000662

RESUMO

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, BAP1 encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes Bcl2 and Mcl1. In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Mitf Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program.


Assuntos
Apoptose/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias Uveais/genética , Animais , Técnicas de Introdução de Genes , Mutação em Linhagem Germinativa , Histonas , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Mesotelioma/genética , Mesotelioma/patologia , Camundongos , Camundongos Mutantes , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitinação , Neoplasias Uveais/patologia
3.
Invest Ophthalmol Vis Sci ; 60(5): 1538-1546, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30994864

RESUMO

Purpose: Recent evidence suggests that retinal photoreceptor cells have an important role in the pathogenesis of retinal microvascular lesions in diabetes. We investigated the role of rod cell phototransduction on the pathogenesis of early diabetic retinopathy (DR) using Gnat1-/- mice (which causes permanent inhibition of phototransduction in rod cells without degeneration). Methods: Retinal thickness, oxidative stress, expression of inflammatory proteins, electroretinograms (ERG) and optokinetic responses, and capillary permeability and degeneration were evaluated at up to 8 months of diabetes. Results: The diabetes-induced degeneration of retinal capillaries was significantly inhibited in the Gnat1-/- diabetics. The effect of the Gnat1 deletion on the diabetes-induced increase in permeability showed a nonuniform accumulation of albumin in the neural retina; the defect was inhibited in diabetic Gnat1-/- mice in the inner plexiform layer (IPL), but neither in the outer plexiform (OPL) nor inner nuclear (INL) layers. In Gnat1-deficient animals, the diabetes-induced increase in expression of inflammatory associated proteins (iNOS and ICAM-1, and phosphorylation of IĸB) in the retina, and the leukocyte mediated killing of retinal endothelial cells were inhibited, however the diabetes-mediated induction of oxidative stress was not inhibited. Conclusions: In conclusion, deletion of transducin1 (and the resulting inhibition of phototransduction in rod cells) inhibits the development of retinal vascular pathology in early DR.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/fisiopatologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Deleção de Genes , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transducina/genética , Visão Ocular/fisiologia , Animais , Permeabilidade Capilar , Retinopatia Diabética/metabolismo , Eletrorretinografia , Proteínas I-kappa B/metabolismo , Immunoblotting , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Óxido Nítrico Sintase Tipo II/metabolismo , Nistagmo Optocinético/fisiologia , Estresse Oxidativo , Fosforilação , Vasos Retinianos/patologia , Estreptozocina , Tomografia de Coerência Óptica
4.
Nat Commun ; 10(1): 1589, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962442

RESUMO

Transmembrane and coiled-coil domains 1 (TMCO1) is a recently identified Ca2+ leak channel in the endoplasmic reticulum. TMCO1 dysfunction in humans is associated with dysmorphism, mental retardation, glaucoma and the occurrence of cancer. Here we show an essential role of TMCO1 in osteogenesis mediated by local Ca2+/CaMKII signaling in osteoblasts. TMCO1 levels were significantly decreased in bone from both osteoporosis patients and bone-loss mouse models. Tmco1-/- mice exhibited loss of bone mass and altered microarchitecture characteristic of osteoporosis. In the absence of TMCO1, decreased HDAC4 phosphorylation resulted in nuclear enrichment of HADC4, which leads to deacetylation and degradation of RUNX2, the master regulator of osteogenesis. We further demonstrate that TMCO1-mediated Ca2+ leak provides local Ca2+ signals to activate the CaMKII-HDAC4-RUNX2 signaling axis. The establishment of TMCO1 as a pivotal player in osteogenesis uncovers a novel potential therapeutic target for ameliorating osteoporosis.


Assuntos
Canais de Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Osteoporose/fisiopatologia , Idoso , Animais , Canais de Cálcio/genética , Sinalização do Cálcio , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pessoa de Meia-Idade , Osteogênese/fisiologia , Osteoporose/metabolismo , Ovariectomia , Estabilidade Proteica , Transdução de Sinais
5.
Mol Brain ; 12(1): 35, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961637

RESUMO

Huntington's disease (HD) is a neurodegenerative disorder caused by a genetic abnormality in the huntingtin gene that leads to a polyglutamine repeat expansion of the huntingtin protein. The cleaved polyglutamine expansion of mutant huntingtin (mHTT) protein can form aggregates strongly correlated with HD progression. We have previously shown that the inhibition of mGluR5 using CTEP, a selective negative allosteric mGluR5 modulator, can delay disease progression and reduce in mHTT aggregates in the zQ175 mouse model of HD. This was paralleled by enhanced catalytic activity of Unc-51-like kinase 1 (ULK1), a kinase modulated by mammalian target of rapamycin (mTOR) and key regulator of autophagy initiation. In the present study, we show that CTEP can correct aberrant phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling detected in zQ175 mice that may underlie the enhanced ULK1 activity and activation of autophagy. We also show that CTEP can facilitate cAMP response element-binding protein (CREB)-mediated expression of brain-derived neurotrophic factor (BDNF) to foster neuronal survival and reduce apoptosis. Taken together, our findings provide the molecular evidence for how targeting mGluR5 using a well-tolerated selective NAM can mitigate two critical mechanisms of neurodegeneration, autophagy and apoptosis.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Regulação Alostérica , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Mutantes , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Phytomedicine ; 59: 152897, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974311

RESUMO

BACKGROUND: We recently reported that alkaloids in Uncaria hook (a constituent of yokukansan) contribute to antagonism of 5-HT3 receptors. Many studies have reported that 5-HT3 receptor antagonists reduce alcohol preference. However, the effect of yokukansan on alcohol preference is not clear. PURPOSE: This study was performed to investigate the direct effect of yokukansan on alcohol preference and the effect of 5-HT3 receptors on the preference. STUDY DESIGN: We examined ethanol preference effected by yokukansan. Next, we analyzed the contribution of 5-HT3 receptors to the effect of yokukansan. METHODS: Ethanol preference was measured using the two-bottle preference test in mice fed with or without yokukansan diet. Next, the contribution of 5-HT3 receptors to ethanol preference was investigated using 5-HT3 receptor-deficient mice. RESULTS: Reduction of ethanol preference by yokukansan was not observed using 5-HT3 receptor deficient mice. CONCLUSION: Yokukansan contributes to reduced ethanol preference and antagonism of 5-HT3 receptors is associated with the effect.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Etanol , Receptores 5-HT3 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Animais , Etanol/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores 5-HT3 de Serotonina/genética
7.
PLoS Genet ; 15(4): e1008034, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017901

RESUMO

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.


Assuntos
Antígenos CD34/metabolismo , Melanócitos/imunologia , Melanócitos/fisiologia , Células-Tronco/imunologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cor de Cabelo/fisiologia , Folículo Piloso/citologia , Folículo Piloso/fisiologia , Melanócitos/classificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Nus , Camundongos Transgênicos , Proteína Básica da Mielina/deficiência , Proteína Básica da Mielina/genética , Crista Neural/citologia , Crista Neural/imunologia , Crista Neural/fisiologia , Pigmentação/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regeneração/fisiologia , Células-Tronco/classificação
8.
Nat Commun ; 10(1): 1431, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926797

RESUMO

Autism spectrum disorder (ASD) is characterized by dysfunction in social interactions, stereotypical behaviours and high co-morbidity with intellectual disability. A variety of syndromic and non-syndromic neurodevelopmental disorders have been connected to alterations in metabotropic glutamate receptor (mGluR) signalling. These receptors contribute to synaptic plasticity, spine maturation and circuit development. Here, we investigate the physiological role of Gprasp2, a gene linked to neurodevelopmental disabilities and involved in the postendocytic sorting of G-protein-coupled receptors. We show that Gprasp2 deletion leads to ASD-like behaviour in mice and alterations in synaptic communication. Manipulating the levels of Gprasp2 bidirectionally modulates the surface availability of mGluR5 and produces alterations in dendritic complexity, spine density and synaptic maturation. Loss of Gprasp2 leads to enhanced hippocampal long-term depression, consistent with facilitated mGluR-dependent activation. These findings demonstrate a role for Gprasp2 in glutamatergic synapses and suggest a possible mechanism by which this gene is linked to neurodevelopmental diseases.


Assuntos
Transtorno Autístico/genética , Transtorno Autístico/psicologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Plasticidade Neuronal/genética , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Comportamento Animal , Espinhas Dendríticas/patologia , Espinhas Dendríticas/ultraestrutura , Deleção de Genes , Hipocampo/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Memória , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Transmissão Sináptica
9.
Lipids Health Dis ; 18(1): 62, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871555

RESUMO

BACKGROUND: Endothelial-to-mesenchymal transition (EndMT) plays significant roles in atherosclerosis, but the regulatory mechanisms involving lncRNAs remain to be elucidated. Here we sort to identify the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in ox-LDL-induced EndMT. METHODS: The atherosclerosis model was established by feeding ApoE-/- mice with high-fat diet, and the levels of lncRNA MALAT1 in mouse arterial tissue were detected by RT-qPCR. Cell model was established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL, and the levels of EndMT markers, such as CD31, vWF, α-SMA and Vimentin and lncRNA MALAT1 levels were detected and their correlations were analyzed. The role of MALAT1 in EndMT and its dependence on Wnt/ß-catenin signaling pathway was further detected by knocking down or overexpressing MALAT1. RESULTS: MALAT1 was upregulated in high-fat food fed ApoE-/- mice. HUVECs treated with ox-LDL showed a significant decrease in expression of CD31 and vWF, a significant increase in expression of α-SMA and vimentin, and upregulated MALAT1. An increased MALAT1 level facilitated the nuclear translocation of ß-catenin induced by ox-LDL. Inhibition of MALAT1 expression reversed nuclear translocation of ß-catenin and EndMT. Moreover, overexpression of MALAT1 enhanced the effects of ox-LDL on HUVEC EndMT and Wnt/ß-catenin signaling activation. CONCLUSIONS: Our study revealed that the pathological EndMT required the activation of the MALAT1-dependent Wnt/ß-catenin signaling pathway, which may be important for the onset of atherosclerosis. TRIAL REGISTRATION: Not applicable.


Assuntos
Aterosclerose/genética , Transição Epitelial-Mesenquimal/genética , RNA Longo não Codificante/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/etiologia , Dieta Hiperlipídica/efeitos adversos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas LDL/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
10.
Mol Brain ; 12(1): 17, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30866987

RESUMO

Hypomyelination in the central nerves system (CNS) is one of the most obviously pathological features in Niemann-Pick Type C disease (NPC), which is a rare neurodegenerative disorder caused by mutations in the NPC intracellular cholesterol transporter 1 or 2 (Npc1 or Npc2). Npc1 plays key roles in both neurons and oligodendrocytes during myelination, however, the linkage between the disturbed cholesterol transport and inhibited myelination is unrevealed. In this study, mass spectrometry (MS)-based differential quantitative proteomics was applied to compare protein composition in the corpus callosum between wild type (WT) and NPC mice. In total, 3009 proteins from both samples were identified, including myelin structural proteins, neuronal proteins, and astrocyte-specific proteins. In line to hypomyelination, our data revealed downregulation of myelin structural and indispensable proteins in Npc1 mutant mice. Notably, the reduced ceramide synthase 2 (Cers2), UDP glycosyltransferase 8 (Ugt8), and glycolipid transfer protein (Gltp) indicate the altered sphingolipid metabolism in the disease and the involvement of Gltp in myelination. The identification of most reported myelin structural proteins and proteins from other cell types advocates the use of the corpus callosum to investigate proteins in different cell types that regulate myelination.


Assuntos
Corpo Caloso/metabolismo , Bainha de Mielina/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteômica , Animais , Ontologia Genética , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Proteínas do Tecido Nervoso/metabolismo
11.
Int Immunopharmacol ; 70: 417-427, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30856392

RESUMO

Fluoroquinolones trigger anaphylaxis during clinical applications, affecting the safety of their administration. Mast cells are immune cells that act as sentinels during host defenses, mediating hypersensitivity and anaphylactic reactions. Mas-related G protein-coupled receptor X2 (MRGPRX2) is a mast cell-specific receptor that mediates cell degranulation in anaphylactic reactions. In this study, the mechanism underpinning the anaphylactic reactions caused by fluoroquinolones was investigated. Hypersensitivity was assessed through hindpaw swelling, tissue fluid leakage assays, in vivo and body temperature measurements assay in vivo, and cell calcium mobilization assays, and mast cell degranulation assays in vitro. Mast cell-deficient W-sash c-kit mutant KitW-sh/W-sh mice and MrgprB2 (the orthologous receptor of MRGPRX2 in mice) knockout mice exhibited reduced fluoroquinolone-induced anaphylactic effects. Fluoroquinolones activated mast cells in a dose-dependent manner and reduced degranulation was observed following MRGPRX2 silencing. These results reveal that fluoroquinolone-induced anaphylactic reactions are mediated by mast cells through MRGPRX2.


Assuntos
Anafilaxia/metabolismo , Fluoroquinolonas/metabolismo , Hipersensibilidade/metabolismo , Mastócitos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Anafilaxia/induzido quimicamente , Anafilaxia/genética , Animais , Temperatura Corporal , Sinalização do Cálcio , Degranulação Celular/genética , Permeabilidade da Membrana Celular/genética , Fluoroquinolonas/química , Células HEK293 , Humanos , Hipersensibilidade/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas-G/genética , Receptores de Neuropeptídeos/genética
12.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923196

RESUMO

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-ß (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.


Assuntos
Tolerância Imunológica , Fagocitose/imunologia , Infecções por Pseudomonas/imunologia , Fagos de Pseudomonas/fisiologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/virologia , Infecção dos Ferimentos/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Anticorpos Antivirais/imunologia , Humanos , Interferons/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fagos de Pseudomonas/imunologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/metabolismo
13.
Science ; 363(6433): 1345-1349, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30898933

RESUMO

Excessive consumption of beverages sweetened with high-fructose corn syrup (HFCS) is associated with obesity and with an increased risk of colorectal cancer. Whether HFCS contributes directly to tumorigenesis is unclear. We investigated the effects of daily oral administration of HFCS in adenomatous polyposis coli (APC) mutant mice, which are predisposed to develop intestinal tumors. The HFCS-treated mice showed a substantial increase in tumor size and tumor grade in the absence of obesity and metabolic syndrome. HFCS increased the concentrations of fructose and glucose in the intestinal lumen and serum, respectively, and the tumors transported both sugars. Within the tumors, fructose was converted to fructose-1-phosphate, leading to activation of glycolysis and increased synthesis of fatty acids that support tumor growth. These mouse studies support the hypothesis that the combination of dietary glucose and fructose, even at a moderate dose, can enhance tumorigenesis.


Assuntos
Carcinogênese/patologia , Dieta/efeitos adversos , Xarope de Milho Rico em Frutose/efeitos adversos , Neoplasias Intestinais/patologia , Carga Tumoral , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Xarope de Milho Rico em Frutose/administração & dosagem , Camundongos , Camundongos Mutantes , Gradação de Tumores
14.
Invest Ophthalmol Vis Sci ; 60(4): 1132-1143, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30901771

RESUMO

Purpose: Bardet-Biedl syndrome (BBS) is an archetypical ciliopathy caused by defective ciliary trafficking and consequent function. Insights gained from BBS mouse models are applicable to other syndromic and nonsyndromic retinal diseases. This progressive characterization of the visual phenotype in three BBS mouse models sets a baseline for testing therapeutic interventions. Methods: Longitudinal acquisition of electroretinograms, optical coherence tomography scans, and visual acuity using the optomotor reflex in Bbs6/Mkks, Bbs8/Ttc8, and Bbs5 knockout mice. Gene and protein expression analysis in vivo and in vitro. Results: Complete loss of BBS5, BBS6, or BBS8 leads to different rates of retinal degeneration and visual function over time. BBS8-deficient mice showed the fastest rate of degeneration, and BBS8 seems to be required for cone photoreceptors to reach functional maturity. In contrast, the loss of BBS5 (a further BBSome component) showed very little degeneration. Loss of BBS8 versus BBS5 resulted in different physiologic responses both in vivo and in vitro. BBS6-deficient mice show a slower rate of degeneration with both rod and cone function reducing at a similar rate. Conclusions: The mouse models analyzed show distinct and diverging courses of degeneration upon loss of BBS5, BBS6, or BBS8, which can be used as a benchmark to test therapeutic interventions. Close consideration of the different phenotypes reveal subtle but important differences relating to their function. Because we also see differences in terms of phenotype depending on the type of visual assessment used, our data highlight the importance of using a combinatorial approach for assessment of visual function.


Assuntos
Síndrome de Bardet-Biedl/fisiopatologia , Modelos Animais de Doenças , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Visão Ocular/fisiologia , Envelhecimento/fisiologia , Animais , Síndrome de Bardet-Biedl/genética , Western Blotting , Proteínas de Transporte/genética , Eletrorretinografia , Técnicas de Genotipagem , Chaperoninas do Grupo II/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/genética , Transdução de Sinais/fisiologia , Tomografia de Coerência Óptica
15.
Science ; 363(6432): 1226-1230, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872527

RESUMO

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.


Assuntos
Síndrome de Noonan/genética , Proteínas Oncogênicas/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Técnicas de Introdução de Genes , Mutação em Linhagem Germinativa , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Camundongos Mutantes , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética
16.
Front Biosci (Landmark Ed) ; 24: 648-687, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844704

RESUMO

Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been associated with any specific physiological function or pathology, and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymatic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiological functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-molecule inhibitors recognizing GCPII/III for diagnosis, imaging and experimental therapy of human cancers and other pathologies.


Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Carboxipeptidases/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Neuropeptídeos/química , Peptídeo Hidrolases/metabolismo , Adenocarcinoma/metabolismo , Animais , Anticorpos Monoclonais/química , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Encéfalo/metabolismo , Modelos Animais de Doenças , Glutamatos/química , Humanos , Hidrólise , Doenças Inflamatórias Intestinais/metabolismo , Intestino Delgado/metabolismo , Jejuno/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Fenótipo , Neoplasias da Próstata/metabolismo , Ratos
17.
Cell Commun Signal ; 17(1): 12, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760304

RESUMO

BACKGROUND: The existence of differentiated thyroid cells is critical to respond radioactive iodide treatment strategy in thyroid cancer, and loss of the differentiated phenotype is a trademark of iodide-refractive thyroid disease. While high-dose therapy has been beneficial to several cancer patients, many studies have indicated this clinical benefit was limited to patients having BRAF mutation. BRAF-targeted paired box gene-8 (PAX8), a thyroid-specific transcription factor, generally dysregulated in BRAF-mutated thyroid cancer. METHODS: In this study, thyroid iodine-metabolizing gene levels were detected in BRAF-transformed thyroid cells after low and high dose of ionizing radiation. Also, an mRNA-targeted approach was used to figure out the underlying mechanism of low (0.01Gyx10 or 0.1Gy) and high (2Gy) radiation function on thyroid cancer cells after BRAFV600E mutation. RESULTS: Low dose radiation (LDR)-induced PAX8 upregulation restores not only BRAF-suppressive sodium/iodide symporter (NIS) expression, one of the major protein necessary for iodine uptake in healthy thyroid, on plasma membrane but also regulate other thyroid metabolizing genes levels. Importantly, LDR-induced PAX8 results in decreased cellular transformation in BRAF-mutated thyroid cells. CONCLUSION: The present findings provide evidence that LDR-induced PAX8 acts as an important regulator for suppression of thyroid carcinogenesis through novel STAT3/miR-330-5p pathway in thyroid cancers.


Assuntos
Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/efeitos da radiação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Glândula Tireoide/patologia , Glândula Tireoide/efeitos da radiação , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Hipotireoidismo/patologia , Iodo/metabolismo , Camundongos Mutantes , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Mutação/genética , Fator de Transcrição PAX8/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/radioterapia , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
18.
In Vitro Cell Dev Biol Anim ; 55(3): 211-219, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30756235

RESUMO

Attenuation of fibroblast growth factor receptor (FGFR) 2b signaling suppresses the differentiation of oral epithelial stem cells to ameloblasts, their survival and viability remaining unaffected; however, its effect on dentin formation is unknown. This study aimed to clarify the effect of attenuation of FGFR2b signaling on odontoblast differentiation and dentin formation. Initially, we used a murine rtTA transactivator/tetracycline promoter system for inducible and reversible attenuation of FGFR2b signaling in adult mice. Experimental animals overexpressed soluble FGFR2b (sFGFR2b), and wild-type controls were selected from the same litter (WT group). Histological analysis of CMV mice confirmed the obliteration of the enamel and ameloblast layer, and micro CT analysis revealed a significant increase in dentin thickness in CMV mice rather than in WT mice (P < 0.05). On analyzing the expression of dentin-related differentiation factors, DSPP, nestin, and OCN were upregulated in CMV mice compared to WT mice after 2 weeks of attenuation of FGFR2b signaling. Thereafter, on overexpressing sFGFR2b in dental pulp stem cells, RUNX2 and ALP were upregulated; however, DSPP, nestin, and OCN were downregulated in CMV mice compared to WT mice. The present results show that attenuation of FGFR2b signaling in the oral epithelium specifically induced odontoblast differentiation and promotes early-stage dentin calcification in dental pulp tissue.


Assuntos
Dentina/crescimento & desenvolvimento , Odontoblastos/citologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Esmalte Dentário/diagnóstico por imagem , Polpa Dentária/citologia , Dentina/metabolismo , Doxiciclina/farmacologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Mutantes , Odontoblastos/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Microtomografia por Raio-X
19.
Nat Med ; 25(4): 583-590, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804514

RESUMO

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1-5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA-target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Mutação com Ganho de Função/genética , MicroRNAs/genética , Animais , Sequência de Bases , Condrócitos/metabolismo , Feminino , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , MicroRNAs/metabolismo , Linhagem , Fenótipo , Transcriptoma/genética
20.
Nat Commun ; 10(1): 838, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783090

RESUMO

Hypoxia is a main driver of sprouting angiogenesis, but how tip endothelial cells are directed to hypoxic regions remains poorly understood. Here, we show that an endothelial MST1-FOXO1 cascade is essential for directional migration of tip cells towards hypoxic regions. In mice, endothelial-specific deletion of either MST1 or FOXO1 leads to the loss of tip cell polarity and subsequent impairment of sprouting angiogenesis. Mechanistically, MST1 is activated by reactive oxygen species (ROS) produced in mitochondria in response to hypoxia, and activated MST1 promotes the nuclear import of FOXO1, thus augmenting its transcriptional regulation of polarity and migration-associated genes. Furthermore, endothelial MST1-FOXO1 cascade is required for revascularization and neovascularization in the oxygen-induced retinopathy model. Together, the results of our study delineate a crucial coupling between extracellular hypoxia and an intracellular ROS-MST1-FOXO1 cascade in establishing endothelial tip cell polarity during sprouting angiogenesis.


Assuntos
Células Endoteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Hipóxia Celular , Polaridade Celular , Células Cultivadas , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neovascularização Patológica/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Espécies Reativas de Oxigênio/metabolismo , Retina/citologia , Retina/fisiologia
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