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1.
Biochem Biophys Res Commun ; 504(4): 857-864, 2018 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-30219233

RESUMO

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor ß (TGF-ß) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-ß is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-ß activation. This study aimed to explore the potential roles of PLK-dependent TGF-ß activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-ß1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-ß activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-ß receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-ß activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-ß activation has potential as a therapeutic strategy for ALI.

2.
Proc Natl Acad Sci U S A ; 115(19): 4969-4974, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29686061

RESUMO

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/prevenção & controle , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Proteína Proto-Oncogênica N-Myc/biossíntese , Células-Tronco Neoplásicas/metabolismo , Tretinoína/análogos & derivados , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Prognóstico , Tretinoína/farmacologia
3.
Sci Rep ; 7(1): 14972, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097807

RESUMO

TDP-43 is an RNA-binding protein important for many aspects of RNA metabolism. Abnormal accumulation of TDP-43 in the cytoplasm of affected neurons is a pathological hallmark of the neurodegenerative diseases frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Several transgenic mouse models have been generated that recapitulate defects in TDP-43 accumulation, thus causing neurodegeneration and behavioural impairments. While aging is the key risk factor for neurodegenerative diseases, the specific effect of aging on phenotypes in TDP-43 transgenic mice has not been investigated. Here, we analyse age-dependent changes in TDP-43 transgenic mice that displayed impaired memory. We found the accumulation of abundant poly-ubiquitinated protein aggregates in the hippocampus of aged TDP-43 transgenic mice. Intriguingly, the aggregates contained some interneuron-specific proteins such as parvalbumin and calretinin, suggesting that GABAergic interneurons were degenerated in these mice. The abundance of aggregates significantly increased with age and with the overexpression of TDP-43. Gene array analyses in the hippocampus and other brain areas revealed dysregulation in genes linked to oxidative stress and neuronal function in TDP-43 transgenic mice. Our results indicate that the interneuron degeneration occurs upon aging, and TDP-43 accelerates age-dependent neuronal degeneration, which may be related to the impaired memory of TDP-43 transgenic mice.

4.
EBioMedicine ; 24: 257-266, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28927749

RESUMO

The acute phase protein orosomucoid-1 (Orm1) is mainly expressed by hepatocytes (HPCs) under stress conditions. However, its specific function is not fully understood. Here, we report a role of Orm1 as an executer of HPC proliferation. Increases in serum levels of Orm1 were observed in patients after surgical resection for liver cancer and in mice undergone partial hepatectomy (PH). Transcriptome study showed that Orm1 became the most abundant in HPCs isolated from regenerating mouse liver tissues after PH. Both in vitro and in vivo siRNA-induced knockdown of Orm1 suppressed proliferation of mouse regenerating HPCs and human hepatic cells. Microarray analysis in regenerating mouse livers revealed that the signaling pathways controlling chromatin replication, especially the minichromosome maintenance protein complex genes were uniformly down-regulated following Orm1 knockdown. These data suggest that Orm1 is induced in response to hepatic injury and executes liver regeneration by activating cell cycle progression in HPCs.


Assuntos
Perfilação da Expressão Gênica/métodos , Hepatócitos/citologia , Neoplasias Hepáticas/cirurgia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Orosomucoide/genética , Animais , Ciclo Celular , Proliferação de Células , Redes Reguladoras de Genes , Hepatectomia , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Regeneração Hepática , Camundongos , Orosomucoide/metabolismo , Transdução de Sinais , Regulação para Cima
5.
Artigo em Inglês | MEDLINE | ID: mdl-26379781

RESUMO

BACKGROUND: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-ß, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-ß is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-ß must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-ß by cleavage between R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R(58) cleavage edges and functions as a footprint of PLK-dependent TGF-ß activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis. RESULTS: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L(59) LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-ß receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L(59) LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) α1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues. CONCLUSIONS: L(59) LAP-DPs reflect PLK-dependent TGF-ß activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.

6.
Anat Rec (Hoboken) ; 295(2): 215-22, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22190445

RESUMO

The Y-box binding protein 1 (YB-1), characterized by the presence of the cold shock domain, has been reported to induce chemoresistance in cancer therapy. Chemotherapy is one of the main therapeutic strategies in the treatment of cancer, in addition to surgery, radiation therapy, and hormonal therapy. However, chemoresistance remains a key obstacle to successful cancer management. In this review, we will focus on the role of YB-1, an important regulator of gene transcription, in cancer therapy and chemoresistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Marcação de Genes , Neoplasias/genética , Proteína 1 de Ligação a Y-Box/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo
7.
J Neurochem ; 110(6): 1965-76, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19645748

RESUMO

Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.


Assuntos
Encéfalo/metabolismo , Síndrome de Down/metabolismo , Síndrome de Down/fisiopatologia , Peroxidação de Lipídeos/fisiologia , Fatores Etários , Aldeídos/metabolismo , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/patologia , Eletroforese em Gel Bidimensional/métodos , Feminino , Regulação da Expressão Gênica/genética , Humanos , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microdiálise , Espécies Reativas de Oxigênio/metabolismo , Trissomia/genética
8.
Hum Mol Genet ; 18(6): 1099-109, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19147686

RESUMO

Mutations in EFHC1 gene have been previously reported in patients with epilepsies, including those with juvenile myoclonic epilepsy. Myoclonin1, also known as mRib72-1, is encoded by the mouse Efhc1 gene. Myoclonin1 is dominantly expressed in embryonic choroid plexus, post-natal ependymal cilia, tracheal cilia and sperm flagella. In this study, we generated viable Efhc1-deficient mice. Most of the mice were normal in outward appearance, and both sexes were found to be fertile. However, the ventricles of the brains were significantly enlarged in the null mutants, but not in the heterozygotes. Although the ciliary structure was found intact, the ciliary beating frequency was significantly reduced in null mutants. In adult stages, both the heterozygous and null mutants developed frequent spontaneous myoclonus. Furthermore, the threshold of seizures induced by pentylenetetrazol was significantly reduced in both heterozygous and null mutants. These observations seem to further suggest that decrease or loss of function of myoclonin1 may be the molecular basis for epilepsies caused by EFHC1 mutations.


Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Predisposição Genética para Doença , Mioclonia/complicações , Mioclonia/genética , Convulsões/complicações , Convulsões/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Ventrículos Cerebrais/metabolismo , Ventrículos Cerebrais/patologia , Cílios/ultraestrutura , Convulsivantes , Giro Denteado/metabolismo , Giro Denteado/patologia , Heterozigoto , Camundongos , Mutação/genética , Mioclonia/patologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Convulsões/patologia , Ácidos Siálicos/metabolismo , Ácido gama-Aminobutírico/metabolismo
9.
Biochem Biophys Res Commun ; 367(1): 226-33, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18164683

RESUMO

EFHC1 is a gene mutated in patients with idiopathic epilepsies, and encodes the myoclonin1 protein. We here report the distribution of myoclonin1 in mouse. Immunohistochemical analyses revealed that the myoclonin1 first appeared at the roof of hindbrain at embryonic day 10 (E10), and moved on to choroid plexus at E14. At E18, it moved to ventricle walls and disappeared from choroid plexus. From neonatal to adult stages, myoclonin1 was concentrated in the cilia of ependymal cells at ventricle walls. At adult stages, myoclonin1 expression was also observed at tracheal epithelial cilia in lung and at sperm flagella in testis. Specificities of these immunohistochemical signals were verified by using Efhc1-deficient mice as negative controls. Results of Efhc1 mRNA in situ hybridization were also consistent with the immunohistochemical observations. Our findings raise "choroid plexusopathy" or "ciliopathy" as intriguing candidate cascades for the molecular pathology of epilepsies caused by the EFHC1 mutations.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Plexo Corióideo/metabolismo , Cílios/metabolismo , Epêndima/metabolismo , Regulação da Expressão Gênica/genética , Adulto , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/embriologia , Epêndima/citologia , Epêndima/embriologia , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Pulmão/citologia , Pulmão/embriologia , Pulmão/metabolismo , Masculino , Camundongos , Mutação , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Testículo/citologia , Testículo/metabolismo , Fatores de Tempo
10.
J Neurosci ; 27(22): 5903-14, 2007 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-17537961

RESUMO

Loss-of-function mutations in human SCN1A gene encoding Nav1.1 are associated with a severe epileptic disorder known as severe myoclonic epilepsy in infancy. Here, we generated and characterized a knock-in mouse line with a loss-of-function nonsense mutation in the Scn1a gene. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the first postnatal month. Immunohistochemical analyses revealed that, in the developing neocortex, Nav1.1 was clustered predominantly at the axon initial segments of parvalbumin-positive (PV) interneurons. In heterozygous knock-in mice, trains of evoked action potentials in these fast-spiking, inhibitory cells exhibited pronounced spike amplitude decrement late in the burst. Our data indicate that Nav1.1 plays critical roles in the spike output from PV interneurons and, furthermore, that the specifically altered function of these inhibitory circuits may contribute to epileptic seizures in the mice.


Assuntos
Axônios/química , Epilepsia/genética , Interneurônios/química , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural , Parvalbuminas/biossíntese , Canais de Sódio/genética , Canais de Sódio/metabolismo , Potenciais de Ação/genética , Animais , Axônios/metabolismo , Linhagem Celular , Epilepsia/metabolismo , Humanos , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Mutantes , Canal de Sódio Disparado por Voltagem NAV1.1 , Rede Nervosa/química , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Inibição Neural/genética , Canais de Sódio/fisiologia
11.
Epilepsia ; 47(10): 1732-6, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17054696

RESUMO

PURPOSE: To investigate the genetic background of familial severe myoclonic epilepsy in infancy (SMEI) cases. METHODS: We performed mutation analyses of the sodium-channel gene SCN1A in two Japanese brothers with clinical features of SMEI and their parents, who had no history of febrile and epileptic seizures. RESULTS: Each patient showed nucleotide changes (c.[730G>T; 735G>T; 736A>T]) in the coding exon 6 of SCN1A that led to a truncation of the channel protein. Their father showed no mutations, but their mother showed the same mutation in a subpopulation of lymphocytes. CONCLUSIONS: The maternal mosaicism explains the identical SCN1A mutations in the two brothers. This highlights the importance of investigating parental mosaicism even in sporadic SMEI cases.


Assuntos
Epilepsias Mioclônicas/genética , Canais Epiteliais de Sódio/genética , Família , Mosaicismo/estatística & dados numéricos , Mutação/genética , Pré-Escolar , Análise Mutacional de DNA , Epilepsias Mioclônicas/diagnóstico , Epilepsias Mioclônicas/epidemiologia , Humanos , Japão/epidemiologia , Masculino , Pais , Irmãos
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