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1.
Proc Natl Acad Sci U S A ; 119(33): e2203632119, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35951651

RESUMO

Epilepsy is a common neurological disorder, which has been linked to mutations or deletions of RNA binding protein, fox-1 homolog (Caenorhabditis elegans) 3 (RBFOX3)/NeuN, a neuronal splicing regulator. However, the mechanism of seizure mediation by RBFOX3 remains unknown. Here, we show that mice with deletion of Rbfox3 in gamma-aminobutyric acid (GABA) ergic neurons exhibit spontaneous seizures and high premature mortality due to increased presynaptic release, postsynaptic potential, neuronal excitability, and synaptic transmission in hippocampal dentate gyrus granule cells (DGGCs). Attenuating early excitatory gamma-aminobutyric acid (GABA) action by administering bumetanide, an inhibitor of early GABA depolarization, rescued premature mortality. Rbfox3 deletion reduced hippocampal expression of vesicle-associated membrane protein 1 (VAMP1), a GABAergic neuron-specific presynaptic protein. Postnatal restoration of VAMP1 rescued premature mortality and neuronal excitability in DGGCs. Furthermore, Rbfox3 deletion in GABAergic neurons showed fewer neuropeptide Y (NPY)-expressing GABAergic neurons. In addition, deletion of Rbfox3 in NPY-expressing GABAergic neurons lowered intrinsic excitability and increased seizure susceptibility. Our results establish RBFOX3 as a critical regulator and possible treatment path for epilepsy.


Assuntos
Proteínas de Ligação a DNA , Neurônios GABAérgicos , Proteínas do Tecido Nervoso , Neuropeptídeo Y , Convulsões , Proteína 1 Associada à Membrana da Vesícula , Animais , Bumetanida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Giro Denteado/metabolismo , Antagonistas GABAérgicos/farmacologia , Neurônios GABAérgicos/metabolismo , Deleção de Genes , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeo Y/metabolismo , Convulsões/genética , Convulsões/metabolismo , Proteína 1 Associada à Membrana da Vesícula/genética , Proteína 1 Associada à Membrana da Vesícula/metabolismo , Ácido gama-Aminobutírico/metabolismo
2.
Hum Mol Genet ; 31(18): 3161-3180, 2022 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-35567414

RESUMO

RTL1/PEG11, which has been associated with anxiety disorders, is a retrotransposon-derived imprinted gene in the placenta. However, imprinting patterns and functions of RTL1 in the brain have not been well-investigated. We found Rtl1 was paternally, but not maternally, expressed in brain stem, thalamus, and hypothalamus of mice, and imprinting status of RTL1 was maintained in human brain. Paternal Rtl1 knockout (Rtl1m+/p-) mice had higher neonatal death rates due to impaired suckling, and low body weights beginning on embryonic day 16.5. High paternal expression of Rtl1 was detected in the locus coeruleus (LC) and Rtl1m+/p- mice showed an increased delay in time of onset for action potentials and inward currents with decreased neuronal excitability of LC neurons. Importantly, Rtl1m+/p- mice exhibited behaviors associated with anxiety, depression, fear-related learning and memory, social dominance, and low locomotor activity. Taken together, our findings demonstrate RTL1 is imprinted in brain, mediates emotional and social behaviors, and regulates excitability in LC neurons.


Assuntos
Proteínas da Gravidez , Retroelementos , Animais , Ansiedade/genética , Transtornos de Ansiedade/genética , Feminino , Impressão Genômica , Humanos , Locus Cerúleo/metabolismo , Camundongos , Neurônios/metabolismo , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Comportamento Social
3.
J Transl Med ; 21(1): 714, 2023 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-37821919

RESUMO

PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.


Assuntos
Metaboloma , Neoplasias da Próstata , Humanos , Masculino , Biópsia , Gradação de Tumores , Antígeno Prostático Específico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/urina , Fatores de Risco , Detecção Precoce de Câncer/métodos , Urinálise/métodos , Urina/química
4.
Hum Mol Genet ; 28(23): 3880-3894, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518394

RESUMO

Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood-brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.


Assuntos
Azóis/administração & dosagem , Doença de Depósito de Glicogênio Tipo II/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Compostos Organosselênicos/administração & dosagem , alfa-Glucosidases/metabolismo , Animais , Azóis/farmacologia , Barreira Hematoencefálica , Encéfalo/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/metabolismo , Gonanos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Injeções Intraperitoneais , Isoindóis , Masculino , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Wortmanina/farmacologia , alfa-Glucosidases/genética
5.
Br J Cancer ; 123(12): 1796-1807, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32963349

RESUMO

BACKGROUND: Breast cancer amplified sequence 2 (BCAS2) plays crucial roles in pre-mRNA splicing and androgen receptor transcription. Previous studies suggested that BCAS2 is involved in double-strand breaks (DSB); therefore, we aimed to characterise its mechanism and role in prostate cancer (PCa). METHODS: Western blotting and immunofluorescence microscopy were used to assay the roles of BCAS2 in the DSBs of PCa cells and apoptosis in Drosophila, respectively. The effect of BCAS2 dosage on non-homologous end joining (NHEJ) and homologous recombination (HR) were assayed by precise end-joining assay and flow cytometry, respectively. Glutathione-S-transferase pulldown and co-immunoprecipitation assays were used to determine whether and how BCAS2 interacts with NBS1. The expression of BCAS2 and other proteins in human PCa was determined by immunohistochemistry. RESULTS: BCAS2 helped repair radiation-induced DSBs efficiently in both human PCa cells and Drosophila. BCAS2 enhanced both NHEJ and HR, possibly by interacting with NBS1, which involved the BCAS2 N-terminus as well as both the NBS1 N- and C-termini. The overexpression of BCAS2 was significantly associated with higher Gleason and pathology grades and shorter survival in patients with PCa. CONCLUSION: BCAS2 promotes two DSB repair pathways by interacting with NBS1, and it may affect PCa progression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Apoptose/genética , DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Drosophila/genética , Humanos , Masculino , Gradação de Tumores , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia
6.
J Biomed Sci ; 27(1): 92, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887585

RESUMO

BACKGROUND: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. METHODS: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. RESULTS: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. CONCLUSIONS: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.


Assuntos
Diferenciação Celular , Variações do Número de Cópias de DNA , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Reprogramação Celular , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taiwan , Adulto Jovem
7.
Br J Cancer ; 120(5): 499-511, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30765871

RESUMO

BACKGROUND: Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. Isolation of a cell-surface protease system for lung cancer metastasis is an important issue for mechanistic studies and therapeutic target identification. METHODS: Immunohistochemistry of a tissue array (n = 64) and TCGA database (n = 255) were employed to assess the correlation between serine protease inhibitors (SPIs) and lung adenocarcinoma progression. The role of SPI in cell motility was examined using transwell assays. Pulldown and LC/MS/MS were performed to identify the SPI-modulated novel protease(s). A xenografted mouse model was harnessed to demonstrate the role of the SPI in lung cancer metastasis. RESULTS: Hepatocyte growth factor activator inhibitor-2 (HAI-2) was identified to be downregulated following lung cancer progression, which was related to poor survival and tumour invasion. We further isolated a serum-derived serine protease, plasmin, to be a novel target of HAI-2. Downregulation of HAI-2 promotes cell surface plasmin activity, EMT, and cell motility. HAI-2 can suppress plasmin-mediated activations of HGF and TGF-ß1, EMT and cell invasion. In addition, downregulated HAI-2 increased metastasis of lung adenocarcinoma via upregulating plasmin activity. CONCLUSION: HAI-2 functions as a novel inhibitor of plasmin to suppress lung cancer cell motility, EMT and metastasis.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Fibrinolisina/metabolismo , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/metabolismo , Células A549 , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/secundário , Animais , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Transição Epitelial-Mesenquimal , Fibrinolisina/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Fator de Crescimento Transformador beta1/metabolismo
8.
Proc Natl Acad Sci U S A ; 112(23): 7243-8, 2015 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-26039995

RESUMO

Signal transducer and activator of transcription 3 (STAT3) had been involved in liver fibrogenesis. We aimed to explore the antifibrotic activities of sorafenib and its derivative SC-1 (devoid of Raf kinase inhibition activity) both in vivo and in vitro with special focus on the STAT3 pathway in hepatic stellate cells (HSCs). The clinical role of STAT3 in chronic hepatitis B (CHB) was also investigated. Experimental fibrosis mouse models were established by thioacetamide injection and bile duct ligation in Balb/C mice and treated with sorafenib and SC-1. Rat and human HSCs were used for mechanistic investigations. Forty CHB patients were enrolled to quantify the hepatic phospho-STAT3 (p-STAT3) levels and correlated with liver fibrosis. Both sorafenib and SC-1 ameliorated liver fibrosis in vivo and promoted HSC apoptosis in vitro. p-STAT3 and downstream signals were down-regulated after sorafenib and SC-1 treatment in HSC. STAT3 overexpression in HSC enhanced cell proliferation and undermined the apoptotic effects of sorafenib and SC-1, whereas STAT3-specific inhibition promoted HSC apoptosis. Sorafenib and SC-1 activated Src-homology protein tyrosine phosphatase-1 (SHP-1) and STAT3 inhibition followed. Of particular interest, in CHB patients with advanced liver fibrosis, p-STAT3 in HSC was significantly overexpressed and positively correlated with the severity of liver fibrosis and plasma IL-6 levels. In conclusion, sorafenib and SC-1 ameliorate liver fibrosis through STAT3 inhibition in HSC and STAT3 may potentially serve as a promising fibrotic biomarker and target in liver fibrosis. SHP-1 phosphatase-directed STAT3 inhibition may represent a previously unidentified strategy for antifibrotic drug discovery.


Assuntos
Cirrose Hepática/prevenção & controle , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Linhagem Celular , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/química , Niacinamida/farmacologia , Compostos de Fenilureia/química , Ratos , Sorafenibe
9.
J Biomed Sci ; 23: 2, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26767504

RESUMO

BACKGROUND: 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages. RESULTS: Human-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 µM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells. CONCLUSION: YC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.


Assuntos
GMP Cíclico/metabolismo , Indazóis/farmacologia , Gotículas Lipídicas/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos
10.
Hepatology ; 59(1): 190-201, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23908138

RESUMO

UNLABELLED: Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma (HCC). Here, we report that Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1) is a major target of sorafenib and generates a series of sorafenib derivatives to search for potent SHP-1 agonists that may act as better anti-HCC agents than sorafenib. Sorafenib increases SHP-1 activity by direct interaction and impairs the association between the N-SH2 domain and the catalytic protein tyrosine phosphatase domain of SHP-1. Deletion of the N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 abolished the effect of sorafenib on SHP-1, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and apoptosis, suggesting that sorafenib may affect SHP-1 by triggering a conformational switch relieving its autoinhibition. Molecular docking of SHP-1/sorafenib complex confirmed our findings in HCC cells. Furthermore, novel sorafenib derivatives SC-43 and SC-40 displayed more potent anti-HCC activity than sorafenib, as measured by enhanced SHP-1 activity, inhibition of p-STAT3, and induction of apoptosis. SC-43 induced substantial apoptosis in sorafenib-resistant cells and showed better survival benefits than sorafenib in orthotopic HCC tumors. CONCLUSION: In this study, we identified SHP-1 as a major target of sorafenib. SC-43 and SC-40, potent SHP-1 agonists, showed better anti-HCC effects than sorafenib in vitro and in vivo. Further clinical investigation is warranted.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Domínio Catalítico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Distribuição Aleatória , Fator de Transcrição STAT3/antagonistas & inibidores , Sorafenibe , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Proc Natl Acad Sci U S A ; 108(45): 18384-9, 2011 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-22042854

RESUMO

Depletion of ß-catenin impairs regeneration of the rapid turn-over gut epithelial cells, but appears dispensable for that of the slow turn-over mature hepatocytes in mice until 1 y of age. As the life span of mature murine hepatocytes is about 400 d, we studied conditional ß-catenin knockout mice (Alb-Cre;Ctnnb1(flx/flx)) until 20 mo of age to determine the function of ß-catenin in the postnatal liver. ß-catenin was absent from the hepatocytes of ß-catenin knockout mice 4 wk after delivery. From 9 mo of age, hepatocytes were gradually replaced by newly formed ß-catenin-positive hepatocytes, which constituted about 90% of hepatocytes at 18-20 mo of age. This process was accompanied by active proliferation of bile duct/ductule cells. ß-catenin-positive hepatocytes exhibited elevated proliferation activity and expression of progenitor cell markers, but lower albumin and Cre. This might explain their intact ß-catenin protein, and suggest their origins from hepatic progenitor cells. Liver tumors arose spontaneously from ß-catenin-positive cells, and tumorigenesis was accelerated by hepatitis B X protein. These results indicate ß-catenin critical for the regeneration of mature hepatocytes. Failure to regenerate mature hepatocytes results in proliferation of hepatic progenitor cells that are able to maintain liver function but are predisposed to form liver tumors.


Assuntos
Hepatócitos/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Células-Tronco/citologia , beta Catenina/metabolismo , Albuminas/genética , Animais , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , beta Catenina/genética
12.
Cell Death Dis ; 15(4): 302, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38684682

RESUMO

Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/ß-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3ß inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.


Assuntos
Glicogênio Sintase Quinase 3 beta , Células-Tronco Pluripotentes Induzidas , Mucopolissacaridose II , Neurônios , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Mucopolissacaridose II/patologia , Mucopolissacaridose II/metabolismo , Mucopolissacaridose II/genética , Diferenciação Celular/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Degeneração Neural/patologia , Cálcio/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-39323088

RESUMO

BACKGROUND: Nuclear receptor interaction protein (NRIP) is versatile and engages with various proteins to execute its diverse biological function. NRIP deficiency was reported to cause small myofibre size in adult muscle regeneration, indicating a crucial role of NRIP in myoblast fusion. METHODS: The colocalization and interaction of NRIP with actin were investigated by immunofluorescence and immunoprecipitation assay, respectively. The participation of NRIP in myoblast fusion was demonstrated by cell fusion assay and time-lapse microscopy. The NRIP mutants were generated for mechanism study in NRIP-null C2C12 (termed KO19) cells and muscle-specific NRIP knockout (NRIP cKO) mice. A GEO profile database was used to analyse NRIP expression in Duchenne muscular dystrophy (DMD) patients. RESULTS: In this study, we found that NRIP directly and reciprocally interacted with actin both in vitro and in cells. Immunofluorescence microscopy showed that the endogenous NRIP colocalized with components of invadosome, such as actin, Tks5, and cortactin, at the tips of cells during C2C12 differentiation. The KO19 cells were generated and exhibited a significant deficit in myoblast fusion compared with wild-type C2C12 cells (3.16% vs. 33.67%, p < 0.005). Overexpressed NRIP in KO19 cells could rescue myotube formation compared with control (3.37% vs. 1.00%, p < 0.01). We further confirmed that NRIP directly participated in cell fusion by using a cell-cell fusion assay. We investigated the mechanism of invadosome formation for myoblast fusion, which depends on NRIP-actin interaction, by analysing NRIP mutants in NRIP-null cells. Loss of actin-binding of NRIP reduced invadosome (enrichment ratio, 1.00 vs. 2.54, p < 0.01) and myotube formation (21.82% vs. 35.71%, p < 0.05) in KO19 cells and forced NRIP expression in KO19 cells and muscle-specific NRIP knockout (NRIP cKO) mice increased myofibre size compared with controls (over 1500 µm2, 61.01% vs. 20.57%, p < 0.001). We also found that the NRIP mRNA level was decreased in DMD patients compared with healthy controls (18 072 vs. 28 289, p < 0.001, N = 10 for both groups). CONCLUSIONS: NRIP is a novel actin-binding protein for invadosome formation to induce myoblast fusion.

14.
World J Mens Health ; 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38863374

RESUMO

PURPOSE: Biomarkers predicting clinically significant prostate cancer (sPC) before biopsy are currently lacking. This study aimed to develop a non-invasive urine test to predict sPC in at-risk men using urinary metabolomic profiles. MATERIALS AND METHODS: Urine samples from 934 at-risk subjects and 268 treatment-naïve PC patients were subjected to liquid chromatography/mass spectrophotometry (LC-MS)-based metabolomics profiling using both C18 and hydrophilic interaction liquid chromatography (HILIC) column analyses. Four models were constructed (training cohort [n=647]) and validated (validation cohort [n=344]) for different purposes. Model I differentiates PC from benign cases. Models II, III, and a Gleason score model (model GS) predict sPC that is defined as National Comprehensive Cancer Network (NCCN)-categorized favorable-intermediate risk group or higher (Model II), unfavorable-intermediate risk group or higher (Model III), and GS ≥7 PC (model GS), respectively. The metabolomic panels and predicting models were constructed using logistic regression and Akaike information criterion. RESULTS: The best metabolomic panels from the HILIC column include 25, 27, 28 and 26 metabolites in Models I, II, III, and GS, respectively, with area under the curve (AUC) values ranging between 0.82 and 0.91 in the training cohort and between 0.77 and 0.86 in the validation cohort. The combination of the metabolomic panels and five baseline clinical factors that include serum prostate-specific antigen, age, family history of PC, previously negative biopsy, and abnormal digital rectal examination results significantly increased AUCs (range 0.88-0.91). At 90% sensitivity (validation cohort), 33%, 34%, 41%, and 36% of unnecessary biopsies were avoided in Models I, II, III, and GS, respectively. The above results were successfully validated using LC-MS with the C18 column. CONCLUSIONS: Urinary metabolomic profiles with baseline clinical factors may accurately predict sPC in men with elevated risk before biopsy.

15.
Hum Mol Genet ; 20(24): 4851-64, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21926084

RESUMO

Pompe disease is caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene, which encodes GAA. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease-induced pluripotent stem cells (PomD-iPSCs) from two patients with different GAA mutations and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to recombinant human GAA reversed the major pathologic phenotypes. Furthermore, l-carnitine treatment reduced defective cellular respiration in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria-related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for the development of novel therapeutic strategies for Pompe disease.


Assuntos
Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Sequência de Bases , Biomarcadores/metabolismo , Carnitina/farmacologia , Carnitina/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Monitoramento de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Doença de Depósito de Glicogênio Tipo II/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , alfa-Glucosidases/farmacologia , alfa-Glucosidases/uso terapêutico
16.
Hepatology ; 56(5): 1913-23, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22505209

RESUMO

UNLABELLED: The liver architecture plays an important role in maintaining hemodynamic balance, but the mechanisms that underlie this role are not fully understood. Hepsin, a type II transmembrane serine protease, is predominantly expressed in the liver, but has no known physiological functions. Here, we report that hemodynamic balance in the liver is regulated through hepsin. Deletion of hepsin (hepsin(-/-) ) in mice resulted in enlarged hepatocytes and narrowed liver sinusoids. Using fluorescent microbeads and antihepsin treatment, we demonstrated that metastatic cancer cells preferentially colonized the hepsin(-/-) mouse liver as a result of the retention of tumor cells because of narrower sinusoids. The enlarged hepatocytes expressed increased levels of connexin, which resulted from defective prohepatocyte growth factor (pro-HGF) processing and decreased c-Met phosphorylation in the livers of hepsin(-/-) mice. Treatment of hepsin(-/-) mice with recombinant HGF rescued these phenotypes, and treatment of wild-type mice with an HGF antagonist recapitulated the phenotypes observed in hepsin(-/-) mice. CONCLUSION: Our findings show that the maintenance of hepatic structural homeostasis occurs through HGF/c-Met/connexin signaling by hepsin, and hepsin-mediated changes in liver architecture significantly enhance tumor metastasis to the liver.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/patologia , Neoplasias Hepáticas/secundário , Fígado/metabolismo , Fígado/patologia , Metástase Neoplásica/patologia , Serina Endopeptidases/metabolismo , Animais , Conexinas/metabolismo , Hemodinâmica , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/genética , Transdução de Sinais
17.
Hepatology ; 55(1): 161-72, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21898507

RESUMO

UNLABELLED: Alteration of cell surface proteolysis has been proposed to play a role in liver fibrosis, a grave complication of biliary atresia (BA). In this study we investigated the roles of hepatocyte growth factor activator inhibitor (HAI)-1 and -2 in the progression of BA. The expression levels of HAI-1 and -2 were significantly increased in BA livers compared with those in neonatal hepatitis and correlated with disease progression. In BA livers, HAI-1 and -2 were coexpressed in cells involved in ductular reactions. In other selective cholangiopathies, ductular cells positive for HAI-1 or HAI-2 also increased in number. Inflammatory cytokines, growth factors, and bile acids differentially up-regulated expression of HAI-1 and -2 transcripts in fetal liver cells and this induction could be antagonized by a cyclooxygenase-2 inhibitor. Conditioned media from cell lines stably overexpressing HAI-1 or HAI-2 enhanced the fibrogenic activity of portal fibroblasts and stellate cells, suggesting that both proteins might be involved in liver fibrosis. Because HAI-1 and -2 colocalized in ductular reactions sharing similar features to those observed during normal liver development, we sought to investigate the role of HAI-1 and -2 in cholangiopathies by exploring their functions in fetal liver cells. Knockdown of HAI-1 or HAI-2 promoted bidirectional differentiation of hepatoblast-derived cells. In addition, we showed that the hepatocyte growth factor activator, mitogen-activated protein kinase kinase 1, and phosphatidylinositol 3-kinase signaling pathways were involved in hepatic differentiation enhanced by HAI-2 knockdown. CONCLUSION: HAI-1 and -2 are overexpressed in the liver in cholangiopathies with ductular reactions and are possibly involved in liver fibrosis and hepatic differentiation; they could be investigated as disease markers and potential therapeutic targets.


Assuntos
Colestase/patologia , Hepatite/patologia , Cirrose Hepática/patologia , Glicoproteínas de Membrana/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Colestase/fisiopatologia , Feminino , Fibroblastos/citologia , Células Estreladas do Fígado/citologia , Hepatite/congênito , Hepatite/fisiopatologia , Hepatócitos/citologia , Humanos , Lactente , Recém-Nascido , Cirrose Hepática/congênito , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Ratos , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia
18.
Haematologica ; 98(5): 729-38, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22983581

RESUMO

The multiple cellular targets affected by proteasome inhibition implicate a potential role for bortezomib, a first-in-class proteasome inhibitor, in enhancing antitumor activities in hematologic malignancies. Here, we examined the antitumor activity and drug targets of bortezomib in leukemia cells. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis assays and associated molecular events assessed by Western Blot. Gene silencing was performed by small interference RNA. Drug was tested in vivo in xenograft models of human leukemia cell lines and in primary leukemia cells. Clinical samples were assessed by immunohistochemical staining. Bortezomib differentially induced apoptosis in leukemia cells that was independent of its proteasome inhibition. Cancerous inhibitor of protein phosphatase 2A, a cellular inhibitor of protein phosphatase 2A, mediated the apoptotic effect of bortezomib. Bortezomib increased protein phosphatase 2A activity in sensitive leukemia cells (HL-60 and KG-1), but not in resistant cells (MOLT-3 and K562). Bortezomib's downregulation of cancerous inhibitor of protein phosphatase 2A and phospho-Akt correlated with its drug sensitivity. Furthermore, cancerous inhibitor of protein phosphatase 2A negatively regulated protein phosphatase 2A activity. Ectopic expression of CIP2A up-regulated phospho-Akt and protected HL-60 cells from bortezomib-induced apoptosis, whereas silencing CIP2A overcame the resistance to bortezomib-induced apoptosis in MOLT3 and K562 cells. Importantly, bortezomib exerted in vivo antitumor activity in HL-60 xenografted tumors and induced cell death in some primary leukemic cells. Cancerous inhibitor of protein phosphatase 2A was expressed in leukemic blasts from bone marrow samples. Cancerous inhibitor of protein phosphatase 2A plays a major role in mediating bortezomib-induced apoptosis in leukemia cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Leucemia/metabolismo , Proteína Fosfatase 2/metabolismo , Pirazinas/farmacologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Bortezomib , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células K562 , Leucemia/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Biol Chem ; 286(38): 33520-32, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21799003

RESUMO

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.


Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Reprogramação Celular , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antígenos de Neoplasias/química , Moléculas de Adesão Celular/química , Claudinas/metabolismo , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Molécula de Adesão da Célula Epitelial , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Estrutura Terciária de Proteína , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
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