Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.111
Filtrar
1.
Adv Exp Med Biol ; 1155: 869-874, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468453

RESUMO

Our group previously reported that taurine has a protective capacity on the hippocampus and cerebellum of arsenic (As)-exposed mouse. In the present study, we explore whether taurine demonstrates protection against As toxicity in primary cortical neurons. Primary cortical neurons were exposed to various concentrations of arsenite and cell viability was assessed to confirm the toxicity of As on cortical neurons. The protection of taurine was examined after primary cortical neurons were treating with arsenite and taurine for 24 h. The cell viability was examined by MTT and caspase-3 activity assay. The expression of Bax and Bcl-2 was determined by western blot. The results showed that As exposure reduced cell viability and enhanced the activity of caspase-3, which were markedly inhibited by taurine treatment. The expression of Bax and Bcl-2 were disturbed by As exposure, which were reversed by taurine. These results indicated that taurine expose protective effect on As-exposed primary cortical neurons and its mechanism maybe involved the regulation of Bax/Bcl-2.


Assuntos
Arsênico/toxicidade , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Taurina/farmacologia , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Camundongos , Neurônios/citologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
2.
Cancer Sci ; 110(9): 2992-3005, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31265190

RESUMO

Cervical clear cell carcinoma (cCCC) constitutes an extremely rare subtype of cervical cancer. Consequently, its pathogenesis remains largely unknown, with no cell lines established from primary tumors. Here, we report the first establishment of cCCC organoids, from biopsy samples of a 23-year-old patient diagnosed with cCCC. By applying a protocol that we recently optimized for gynecological tumors, we were able to propagate a patient-derived cell line (PDC) for more than 6 months as organoids. This PDC tolerated cryopreservation and proliferated either as spheroids or adherent cells, and developed xenografts in immunodeficient mice, ensuring robust utility as a cell line. Intriguingly, the resected tumor focally contained serous carcinoma (SC) in a tiny protruding lesion. Both organoids and derivative xenografts resembled the CCC component of the original tumor in histology, immunostaining profile, and genome-wide copy number changes, including focal gain of MET. Genomic analysis revealed that both organoids and the CCC component harbored only a few mutations, of which 2 mutations were shared in common. In contrast, the SC component showed a mutator-phenotype and prominent genome instability along with biallelic inactivation of TP53, but none of them were found in organoids or the CCC component. The PDC proved sensitive to major chemotherapeutic agents and MET inhibitors. These observations clearly indicated that the PDC, designated as YMC7, can be used as a novel cCCC cell line and provide novel insights into the pathogenesis of mixed cervical adenocarcinoma. As a valuable resource for rare cancer, it will likely contribute to investigations in many fields.


Assuntos
Adenocarcinoma de Células Claras/patologia , Colo do Útero/patologia , Organoides , Cultura Primária de Células , Neoplasias do Colo do Útero/patologia , Adenocarcinoma de Células Claras/cirurgia , Adulto , Animais , Linhagem Celular Tumoral , Colo do Útero/citologia , Colo do Útero/cirurgia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Esferoides Celulares , Neoplasias do Colo do Útero/cirurgia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
3.
Cancer Sci ; 110(9): 2748-2759, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301081

RESUMO

In hepatocarcinogenesis induced by diethylnitrosamine (DEN) in B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. The livers of transgenic mice with a hepatocyte-specific human BRAFV600E mutation weighed 4.5 times more than that of normal mice and consisted entirely of hepatocytes, resembling DEN-induced preneoplastic hepatocytes. However, these transgenic mice spontaneously died 7 wk after birth, therefore this study aimed to clarify the causes of death. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis and thrombocytosis, and activated platelets were deposited in hepatic sinusoids. TPO was also overexpressed in the DEN-induced hepatic tumors, and sinusoidal platelet deposition was observed in the hepatic tumors of humans and mice. Podoplanin was expressed in some of the Kupffer cells in the liver of the transgenic mice, indicating that platelet activation occurred via the interaction of podoplanin with C-type lectin receptor 2 (CLEC-2) on the platelet membrane. Additionally, erythrocyte dyscrasia and glomerulonephropathy/interstitial pneumonia associated with platelet deposition were observed. In the transgenic mice, aspirin (Asp) administration prevented platelet activation, reduced the liver/body weight ratio, decreased the platelet deposition in the liver, kidney, and lung, and prevented erythrocyte dyscrasia and ameliorated the renal/pulmonary changes. Thrombopoietin overproduction by BRAFV600E-mutated hepatocytes may contribute to hepatocyte proliferation via thrombocytosis, platelet activation, and the interaction of platelets with hepatic sinusoidal cells, while hematologic, renal, and pulmonary disorders due to aberrant platelet activation may lead to spontaneous death in the transgenic mice.


Assuntos
Carcinogênese/genética , Neoplasias Hepáticas Experimentais/patologia , Fígado/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Trombopoetina/metabolismo , Animais , Biópsia , Plaquetas/patologia , Medula Óssea/patologia , Capilares/patologia , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Proliferação de Células/genética , Dietilnitrosamina/administração & dosagem , Dietilnitrosamina/toxicidade , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatectomia , Hepatócitos/patologia , Humanos , Fígado/irrigação sanguínea , Fígado/citologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Ativação Plaquetária/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Tumorais Cultivadas
4.
Life Sci ; 231: 116674, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31344427

RESUMO

Hypertrophic scar formation is a fibroproliferative disorder caused by abnormal wound healing. At present, there are limited treatment strategies for hypertrophic scars. In this study, we identified an endogenous peptide, LYENRL, through peptidomics screening that is downregulated in scar skin tissues. The peptide exhibited concentration dependent inhibitory effects on the proliferation, migration and extracellular matrix (ECM) production of scar fibroblasts. By eukaryotic transcriptome sequencing analysis, we noted that LYENRL downregulated gene sets in scar fibroblasts were associated with the transforming growth factor-ß (TGF-ß) signaling pathway. Further experiments revealed that LYENRL was able to inhibit the activation of TGF-ß1/Smad signaling and TGF-ß1-induced activation of scar fibroblasts at the source by blocking the binding of AP-1 to the corresponding region of the Tgfb1 promoter, which in turn inhibited gene expression of Tgfb1. Taken together, we concluded that the effects of LYENRL on scar fibroblasts make it a potential peptide drug for hypertrophic scar treatment.


Assuntos
Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patologia , Peptídeos/farmacologia , Actinas/metabolismo , Linhagem Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Peptídeos/metabolismo , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Pele/metabolismo , Proteínas Smad/metabolismo , Proteínas Smad/fisiologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/fisiologia
5.
Nat Commun ; 10(1): 3004, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285436

RESUMO

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.


Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Rabdomiossarcoma/genética , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Rabdomiossarcoma/patologia , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos
6.
Nat Commun ; 10(1): 2966, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273213

RESUMO

Mutations in genes encoding components of BAF (BRG1/BRM-associated factor) chromatin remodeling complexes cause neurodevelopmental disorders and tumors. The mechanisms leading to the development of these two disease entities alone or in combination remain unclear. We generated mice with a heterozygous nervous system-specific partial loss-of-function mutation in a BAF core component gene, Smarcb1. These Smarcb1 mutant mice show various brain midline abnormalities that are also found in individuals with Coffin-Siris syndrome (CSS) caused by SMARCB1, SMARCE1, and ARID1B mutations and in SMARCB1-related intellectual disability (ID) with choroid plexus hyperplasia (CPH). Analyses of the Smarcb1 mutant animals indicate that one prominent midline abnormality, corpus callosum agenesis, is due to midline glia aberrations. Our results establish a novel role of Smarcb1 in the development of the brain midline and have important clinical implications for BAF complex-related ID/neurodevelopmental disorders.


Assuntos
Anormalidades Múltiplas/genética , Agenesia do Corpo Caloso/genética , Corpo Caloso/crescimento & desenvolvimento , Face/anormalidades , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Micrognatismo/genética , Pescoço/anormalidades , Proteína SMARCB1/genética , Anormalidades Múltiplas/diagnóstico por imagem , Agenesia do Corpo Caloso/diagnóstico por imagem , Agenesia do Corpo Caloso/patologia , Alelos , Animais , Criança , Pré-Escolar , Corpo Caloso/citologia , Corpo Caloso/diagnóstico por imagem , Modelos Animais de Doenças , Embrião de Mamíferos , Face/diagnóstico por imagem , Feminino , Deformidades Congênitas da Mão/diagnóstico por imagem , Humanos , Lactente , Deficiência Intelectual/diagnóstico por imagem , Mutação com Perda de Função , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Micrognatismo/diagnóstico por imagem , Pescoço/diagnóstico por imagem , Neuroglia/patologia , Cultura Primária de Células
7.
Nat Commun ; 10(1): 2987, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278260

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFß1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.


Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Lipogênese/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Metformina/farmacologia , Miofibroblastos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Modelos Animais de Doenças , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Pulmão/citologia , Pulmão/patologia , Masculino , Metformina/uso terapêutico , Camundongos , Miofibroblastos/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos
8.
Nat Commun ; 10(1): 3028, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292434

RESUMO

Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications.


Assuntos
Ataxia Cerebelar/genética , Córtex Cerebelar/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Caseína Quinase Idelta , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Ataxia Cerebelar/patologia , Córtex Cerebelar/citologia , Córtex Cerebelar/patologia , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Proteínas Nucleares/genética , Fosforilação/fisiologia , Cultura Primária de Células , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
9.
Nat Commun ; 10(1): 3039, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292446

RESUMO

TFF1, a secreted protein, plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia, high-grade dysplasia, and adenocarcinomas. In this study, we demonstrate nuclear localization of p-STATY705, with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity, suppressing expression of its target genes. TFF1 blocks IL6Rα-GP130 complex formation through interfering with binding of IL6 to its receptor IL6Rα. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis.


Assuntos
Adenocarcinoma/genética , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Fator Trefoil-1/genética , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células Epiteliais , Mucosa Gástrica/citologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células/métodos , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/patologia , Análise Serial de Tecidos , Fator Trefoil-1/metabolismo
10.
Life Sci ; 231: 116517, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31150684

RESUMO

Our previous study indicated that microRNA 145 (miR-145) and its predicated target, erythropoietin-producing hepatoma (EPH) receptor A4 (EPHA4), was closely associated with ischemic stroke. In this study, we aimed to further explore their function in a model of oxygen-glucose deprivation (OGD). The expression of miR-145 in the blood of 44 patients with ischemic stroke and 37 normal controls was detected by qRT-PCR. After transfection with either the wild- or mutant-type pGL3-promoter EPHA4 3'UTR into the miR-145 mimic and miR-145 inhibitor, a dual-luciferase reporter assay was performed to explore the interaction between miR-145 and EPHA4. qRT-PCR and Western blot were performed to further explore the effects of miR-145 on EPHA4 expression after an miR-145 mimic, an miR-145 inhibitor or LV-sh-EPHA4 was transfected into cerebral cortical neurons. The expression of miR-145 was significantly upregulated in the blood of patients with ischemic stroke compared to that of normal controls. Dual-luciferase reporter assay, qRT-PCR and Western blot results indicated that miR-145 indeed targets EPHA4 through its 3'-UTR and regulates the expression level of EPHA4 at both the mRNA and protein levels. Moreover, the OGD model was successfully constructed, and miR-145 exerted a protective effects in cell viability in the OGD model by downregulating EPHA4. The expression of LOC105376244 could be regulated by the miR-145-EPHA4 interaction. MiR-145 exerted a protective effects in cell viability in the OGD model by downregulating EPHA4, which suggested their potential roles in ischemic stroke and requires further research.


Assuntos
Isquemia Encefálica/metabolismo , Córtex Cerebral/citologia , MicroRNAs/genética , Neurônios/citologia , Receptor EphA4/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Isquemia Encefálica/patologia , Hipóxia Celular/fisiologia , Sobrevivência Celular/genética , Córtex Cerebral/metabolismo , Regulação para Baixo , Feminino , Glucose/metabolismo , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Neurônios/metabolismo , Oxigênio/metabolismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , Acidente Vascular Cerebral/metabolismo
11.
Nat Commun ; 10(1): 2588, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197172

RESUMO

The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.


Assuntos
Aneuploidia , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Segregação de Cromossomos/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Instabilidade Genômica , Humanos , Cinetocoros/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Cultura Primária de Células , Deleção de Sequência , Fuso Acromático/metabolismo
12.
Scand J Immunol ; 90(4): e12800, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31241785

RESUMO

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations, there has been less progress with developing treatment strategies for microcystic malformations. In this study, we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures, we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development.


Assuntos
Linfonodos/crescimento & desenvolvimento , Anormalidades Linfáticas/genética , Vasos Linfáticos/patologia , Transcriptoma , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Linfonodos/embriologia , Cultura Primária de Células , Ligação Proteica , Análise Serial de Tecidos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rho de Ligação ao GTP/genética
13.
J Biol Regul Homeost Agents ; 33(3): 675-685, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189490

RESUMO

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.


Assuntos
Proteína C-Reativa/genética , Moléculas de Adesão Celular/genética , Endométrio/citologia , Células Epiteliais/citologia , Hialuronan Sintases/genética , Componente Amiloide P Sérico/genética , Animais , Proliferação de Células , Feminino , Cultura Primária de Células , Suínos
14.
J Biol Regul Homeost Agents ; 33(3): 695-706, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31184088

RESUMO

Lipids are an alternative energy source for cells and provide structural integrity in cell membrane and their metabolism is regulated with the use of different pathways, such as integrin signalling, oxidative stress, mechanical stress, and pH changes. All of those processes take place in the oral mucosa which is subject to different environmental impacts. In this study, porcine buccal pouch mucosal cells (pBPMCs) were used during long-term primary in vitro culture. The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation. In the results of the following microarray analysis, we analyzed the genes detected, belonging to ontology groups, such as "cellular lipid metabolic process", "response to lipid" and "response to lipopolysaccharides. All of the genes involved in these ontological groups were expressed at higher levels at 7 days of IVC and substantially decreased in expression at days 15 and 30 of primary culture. We observed new genes, which may be recognized as markers in regulation of lipid metabolism in mucosal cells in vitro. The results suggested that the biochemical mechanism-involved lipids were accompanied by increased enzymatic activation and synthesis of crucial growth factors reaching high activity at day 7 of culture, which is also well documented as a stage of tissue regeneration period within oral mucosa. Therefore, this "biochemical fingerprint" may be an additional checkpoint of the integrity, resistance and easy adaptability of oral tissues, which are important conditions of success in tissue engineering and grafting for tissue reconstruction.


Assuntos
Expressão Gênica , Metabolismo dos Lipídeos , Lipopolissacarídeos , Mucosa Bucal/citologia , Animais , Células Cultivadas , Bochecha , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Suínos
15.
J Dairy Sci ; 102(8): 7522-7535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155243

RESUMO

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.


Assuntos
Bovinos/genética , Hepatócitos/metabolismo , Fígado/citologia , Receptores da Somatotropina/genética , Animais , Bovinos/metabolismo , Células Cultivadas , Dexametasona/farmacologia , Feminino , Hormônio do Crescimento/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Gravidez , Cultura Primária de Células , Receptores da Somatotropina/metabolismo , Vimentina/genética , Vimentina/metabolismo
16.
Life Sci ; 230: 197-207, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31150688

RESUMO

AIMS: Increased amounts of protein, in particular albumin within renal tubular cells (TBCs), induce the expression of inflammatory and fibrogenic mediators, which are adverse prognostic factors in tubulointerstitial fibrosis and diabetic nephropathy (DN). We sought to assess the participation of the thiol-linked tertiary structure of albumin in the mechanism of protein toxicity in a model of TBCs. MATERIALS AND METHODS: Cultured human renal proximal tubular cells, HK-2, were exposed to isolated albumin from patients with and without DN (Stages 0, 1 and 4). The magnitude of change of the albumin tertiary structure, cell viability (LDH leakage), apoptosis (Annexin V), transdifferentiation and reticulum endoplasmic stress (Western blot and flow cytometry) and lysosomal enzyme activity were assessed. KEY FINDINGS: We found that albumin from Stage 4 patients presented >50% higher thiol-dependent changes of tertiary structure compared to Stages 0 and 1. Cells incubated with Stage 4 albumin displayed 5 times less viability, accompanied by an increased number of apoptotic cells; evidence of profibrogenic markers E-cadherin and vimentin and higher expression of epithelial-to-mesenchymal transition markers α-SMA and E-cadherin and of endoplasmic reticulum stress protein GRP78 were likewise observed. Moreover, we found that cathepsin B activity in isolated lysosomes showed a significant inhibitory effect on albumin from patients in advanced stages of DN and on albumin that was intentionally modified. SIGNIFICANCE: Overall, this study showed that thiol-dependent changes in albumin's tertiary structure interfere with the lysosomal proteolysis of renal TBCs, inducing molecular changes associated with interstitial fibrosis and DN progression.


Assuntos
Nefropatias Diabéticas/metabolismo , Lisossomos/fisiologia , Albumina Sérica Humana/fisiologia , Adulto , Idoso , Albuminas/metabolismo , Apoptose/efeitos dos fármacos , Caderinas/metabolismo , Linhagem Celular , Sobrevivência Celular , Transdiferenciação Celular , Nefropatias Diabéticas/fisiopatologia , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fibrose , Humanos , Túbulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Estrutura Terciária de Proteína/fisiologia , Albumina Sérica Humana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vimentina/metabolismo
17.
Nat Commun ; 10(1): 2563, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189882

RESUMO

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.


Assuntos
Ritmo Circadiano/genética , Elementos E-Box/genética , Proteínas Circadianas Period/genética , Regiões Promotoras Genéticas/genética , Animais , Comportamento Animal/fisiologia , Temperatura Corporal/fisiologia , Células Cultivadas , Fibroblastos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Cultura Primária de Células , RNA Mensageiro/metabolismo
18.
Nat Commun ; 10(1): 2572, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189917

RESUMO

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/fisiologia , Chaperonas Moleculares/metabolismo , Ataxias Espinocerebelares/patologia , Animais , Retroalimentação Fisiológica/efeitos dos fármacos , Fibroblastos , Células HEK293 , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Hipocampo/patologia , Holoenzimas/metabolismo , Humanos , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ataxias Espinocerebelares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
19.
Nat Commun ; 10(1): 2730, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227691

RESUMO

Recently our groups discovered lugdunin, a new cyclic peptide antibiotic that inhibits Staphylococcus aureus epithelial colonization in humans and rodents. In this work, we analyzed its immuno-modulatory and antimicrobial potential as a single agent or in combination with other microbiota- or host-derived factors. We show that pretreatment of primary human keratinocytes or mouse skin with lugdunin in combination with microbiota-derived factors results in a significant reduction of S. aureus colonization. Moreover, lugdunin increases expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin, and results in the recruitment of monocytes and neutrophils in vivo, both by a TLR/MyD88-dependent mechanism. Interestingly, S. aureus elimination by lugdunin is additionally achieved by synergistic antimicrobial activity with LL-37 and dermcidin-derived peptides. In summary, our results indicate that lugdunin provides multi-level protection against S. aureus and may thus become a promising treatment option for S. aureus skin infections in the future.


Assuntos
Antibacterianos/farmacologia , Imunidade Inata/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Tiazolidinas/farmacologia , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Microbiota/imunologia , Peptídeos/imunologia , Peptídeos Cíclicos/uso terapêutico , Cultura Primária de Células , Pele/efeitos dos fármacos , Pele/imunologia , Pele/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Tiazolidinas/uso terapêutico
20.
Nat Commun ; 10(1): 2757, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227697

RESUMO

Inappropriate expansion of the adipose cells in the subcutaneous adipose tissue (SAT) is a characteristic of hypertrophic obesity and of individuals with genetic predisposition for T2D (first-degree relatives; FDR). It is associated with insulin resistance, a dysfunctional, adipose tissue and reduced adipogenesis. We examined the regulation of adipogenesis in human SAT precursor cells and found ZNF521 to be a critical regulator of early adipogenic commitment and precursor cells leaving the cell cycle. However, neither altered upstream signalling nor lack of SAT progenitor cells could explain the reduced adipogenesis in hypertrophic obesity. Instead, we show that progenitor cells undergoing poor differentiation are characterized by senescence, inability to suppress p53/P16INK4 and secretion of factors reducing adipogenesis in non-senescent cells. We found aging, FDR and established T2D to be associated with increased progenitor cell senescence, reduced adipogenesis and hypertrophic expansion of the SAT adipose cells.


Assuntos
Adipogenia , Senescência Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Obesidade/patologia , Gordura Subcutânea/patologia , Adipócitos , Adulto , Idoso , Envelhecimento/fisiologia , Biópsia por Agulha , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Feminino , Predisposição Genética para Doença , Humanos , Hipertrofia/patologia , Masculino , Pessoa de Meia-Idade , Obesidade/etiologia , Cultura Primária de Células , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Gordura Subcutânea/citologia , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA