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1.
Tumour Biol ; 42(9): 1010428320954735, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32873193

RESUMO

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.


Assuntos
Ambystoma mexicanum , Proliferação de Células/efeitos dos fármacos , Misturas Complexas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-myc/genética
2.
Hum Cell ; 33(3): 590-598, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32474770

RESUMO

Cell lines are powerful tools for research into liver function at the molecular level. However, they are generally unsuitable for rigorously assessing the effects of amino acid composition, because many lines require serum-containing medium for their maintenance. Here, we aimed to investigate the effects of ornithine and arginine, which are included in the characteristic metabolic process in hepatocyte, on a human hepatoma-derived cell line (FLC-4) that can be cultured in serum-free medium. FLC-4 cells were cultured under the following three conditions: + ornithine/ - arginine, - ornithine/ - arginine, and -ornithine/ + arginine. Albumin expression evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay and showed no obvious differences based on the presence of ornithine or arginine. However, the mRNA levels of two liver-enriched transcription factors (CEBPB and HNF1A), which are involved in regulating albumin expression, were significantly higher in cells grown in medium-containing arginine than that in cells grown in ornithine-containing medium. Western blotting showed that the levels both activating and inhibitory C/EBPß isoforms were significantly increased in cells grown in arginine medium. Furthermore, we have found that depletion of both ornithine and arginine, the polyamine sources, in the medium did not cause polyamine deficiency. When ornithine and arginine were depleted, albumin production was significantly reduced at the mRNA level, CEBPB mRNA levels were increased, and the level of activating form of C/EBPß was increased. The results of this study suggest that in hepatocyte, these two amino acids might have different functions, and because of which they elicit disparate cellular responses.


Assuntos
Aminoácidos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/genética , Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/genética , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismo , Arginina/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Meios de Cultura , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Ornitina/farmacologia , RNA Mensageiro/metabolismo
3.
EBioMedicine ; 52: 102635, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32028069

RESUMO

BACKGROUND: The ovulatory dysfunction mechanisms underlying polycystic ovary syndrome (PCOS) are not completely understood. There is no effective therapy for PCOS so far. METHODS: We measured the expression of four and a half LIM domain 2 (FHL2) and other related-genes in human granulosa cells (hGCs) from patients with and without PCOS. To minimise the heterogeneity of patients with PCOS, we only included PCOS patients meeting all three criteria according to the revised Rotterdam consensus. The in vitro effects of FHL2 on ovulatory genes and the underlying mechanisms were examined in KGN cells. The role of FHL2 in ovulation was investigated in vivo by overexpressing FHL2 in rat ovaries via intrabursal lentivirus injection. FINDINGS: Increased FHL2 and androgen receptor (AR) expression and decreased CCAAT/enhancer-binding protein ß (C/EBPß) expression were observed in hGCs from patients with PCOS. FHL2 inhibited the expression of ovulation-related genes, including phosphorylated ERK1/2, C/EBPß, COX2 and HAS2 in KGN cells. It was partially by interacting with AR to act as its co-regulator to inhibit C/EBPß expression and by binding to ERK1/2 to inhibit its phosphorylation. Moreover, FHL2 abundance in hGCs was positively correlated with the basal serum testosterone concentration of patients with PCOS, and dihydrotestosterone (DHT)-induced FHL2 upregulation was mediated by AR signalling in KGN cells. Additionally, lentiviral-mediated functional FHL2 overexpression in rat ovaries for 1 week contributed to an impaired superovulatory response, displaying decreased numbers of retrieved oocytes and a lower MII oocyte rate. 3-week FHL2 overexpression rat models without superovulation led to acyclicity and polycystic ovary morphology. INTERPRETATION: Our findings provide novel insights into the mechanisms underlying the pathogenesis of PCOS, suggesting that FHL2 could be a potential treatment target for ovulatory obstacles in PCOS. FUND: National Key Research and Development Program of China, National Natural Science Foundation, National Institutes of Health project and Shanghai Commission of Science and Technology.


Assuntos
Regulação da Expressão Gênica , Proteínas com Homeodomínio LIM/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Ovulação/genética , Síndrome do Ovário Policístico/etiologia , Síndrome do Ovário Policístico/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/genética , Animais , Biomarcadores , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Modelos Animais de Doenças , Feminino , Imunofluorescência , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Musculares/metabolismo , Ligação Proteica , Ratos , Receptores Androgênicos/genética , Fatores de Transcrição/metabolismo
4.
J Steroid Biochem Mol Biol ; 199: 105608, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31996328

RESUMO

Ovarian granulosa cells, known to be endocrine cells, have well active TLR4-/NFKB signalling mediated innate immune capabilities. We have previously shown that endotoxin not only transiently regulates proinflammatory cytokines but cells become tolerant on repeated exposure to endotoxin and impaired granulosa cells functions, which includes downregulation of CYP19A1 gene. To understand further endotoxin tolerance and impaired granulosa cells function, genome-wide transcriptomic profiling in endotoxin tolerant buffalo granulosa cells (bGCs) identified miR-326 as upregulated amongst top 5 DE miRNAs [unpublished data] and qPCR validation confirmed its upregulation during endotoxin tolerance. In silico analyses showed that miR-326 targets CYP19A1 gene. Therefore, in the present study, we elucidated the role of miR-326 in buffalo granulosa cells (bGCs). We first validated its expression vis-à-vis CYP19A1 gene expression in bGCs, both in vivo and in vitro. Results showed an inverse relationship between miR-326 and CYP19A1 expression. Similarly, transcription factors, known to be involved in CYP19A1 gene regulation, CREB and C/EBP-ß expression was also found to be decreased in granulosa cells mimicking pre-ovulatory follicular stage. Further, miR-326 mimic was transfected to bGCs in culture and expression of CYP19A1 and CREB & C/EBP-ß and genes encoding other enzymes of steroidogenesis pathway were also analyzed. The present study results showed that miR-326 significantly inhibits the expression of CYP19A1 gene while expression of transcription factors CREB and C/EBP-ß was found to be upregulated. The expression of STAR and CYP11A1 was found to be unaffected. To elucidate the molecular mechanism of miR-326 mediated downregulation of CYP19A1, binding analyses of RNA polymerase II and CEBP-ß to CYP19A1 gene promoter II was analyzed. The result also showed decreased binding of RNA polymerase II with increased binding of CEBP-ß to CYP19A1 gene promoter II in bGCs, transfected with miR-326 as compared to control. In summary, our results suggest that miR-326 upregulate CREB and CREB may activate C/EBP-ß and later inhibited the transcription of CYP19A1 and decreased estradiol-17b production. The miR-326 mediated down-regulation of the CYP19A1 gene involving CREB-C/EBP-ß can be exploited in developing strategies to attenuate endotoxin-mediated tolerance induced impaired granulosa cells function to ensure proper fertility in females.


Assuntos
Aromatase/genética , Estradiol/genética , Estrogênios/genética , MicroRNAs/genética , Animais , Búfalos/genética , Búfalos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Bovinos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Estradiol/biossíntese , Estrogênios/biossíntese , Feminino , Regulação da Expressão Gênica/genética , Células da Granulosa/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais
5.
Mol Cell Biochem ; 463(1-2): 211-223, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31686316

RESUMO

Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPß) is a transcription factor, and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPß regulates inflammation, endoplasmic reticulum (ER) stress, and apoptosis, in macrophage foam cells and its underlying molecular mechanism remains unknown. Here, we investigated the effect of C/EBPß knockdown on proteins and genes implicated in inflammation, ER stress, apoptosis, and autophagy in macrophage foam cells. RAW264.7 macrophage cells were transfected with control and C/EBPß-siRNA and then treated with nLDL and oxLDL. Key proteins and genes involved in inflammation, ER stress, apoptosis, and autophagy were analyzed by western blot and qPCR. We found that short interfering RNA (siRNA)-mediated knockdown of C/EBPß attenuated atherogenic lipid-mediated induction of proteins and genes implicated in inflammation (P-NFkB-p65, NFkB-p65, and TNFα), ER stress (ATF4 and ATF6), and apoptosis (CHOP, caspase 1, 3, and 12). Interestingly, C/EBPß knockdown upregulated the expression of autophagy proteins (LC3A/B-II, ATG5) and genes (LC3B, ATG5) but decreased the mammalian target of rapamycin (mTOR) protein phosphorylation and mTORC1 gene expression in oxLDL-loaded RAW264.7 macrophage cells. More importantly, treatment with rapamycin (inhibitor of mTOR) increased expression of proteins implicated in autophagy and cholesterol efflux in oxLDL-loaded RAW 264.7 macrophage cells. The present results suggest that C/EBPß inactivation regulates macrophage foam cell formation in atherogenesis by reducing inflammation, ER stress, and apoptosis and by promoting autophagy and inactivating mTOR.


Assuntos
Apoptose , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Estresse do Retículo Endoplasmático , Células Espumosas/metabolismo , Regulação da Expressão Gênica , Lipoproteínas LDL/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Células Espumosas/patologia , Técnicas de Silenciamento de Genes , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipoproteínas LDL/genética , Camundongos , Células RAW 264.7
6.
BMC Genomics ; 20(1): 878, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747871

RESUMO

BACKGROUND: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. RESULTS: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. CONCLUSION: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.


Assuntos
Doenças dos Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/veterinária , Receptor 2 Toll-Like/genética , Peixe-Zebra/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Quimiocina CXCL11/genética , Quimiocina CXCL11/imunologia , Resistência à Doença/genética , Embrião não Mamífero , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/microbiologia , Linfotoxina-alfa/genética , Linfotoxina-alfa/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Fatores de Transcrição Maf/genética , Fatores de Transcrição Maf/imunologia , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/imunologia , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/imunologia , Mycobacterium marinum/patogenicidade , Neutrófilos/imunologia , Neutrófilos/microbiologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/imunologia , Transcriptoma/imunologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia
7.
BMC Res Notes ; 12(1): 717, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672165

RESUMO

OBJECTIVE: CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPß expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPß, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPß, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPß, the band was present when C/EBPß was knocked down, confirming specificity for a protein other than C/EBPß. The objective of this work was to identify the contaminating reactivity. RESULTS: We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPß is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPß isoforms.


Assuntos
Anticorpos Monoclonais/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Diferenciação Celular/imunologia , Mioblastos/imunologia , Animais , Especificidade de Anticorpos/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Reações Cruzadas/imunologia , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/imunologia , Mioblastos/citologia , Mioblastos/metabolismo , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/imunologia , Cadeias Leves de Miosina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Espectrometria de Massas em Tandem/métodos
8.
PLoS One ; 14(10): e0222717, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31603915

RESUMO

INTRODUCTION: The subventricular zone (SVZ) in the brain is associated with gliomagenesis and resistance to treatment in glioblastoma. In this study, we investigate the prognostic role and biological characteristics of subventricular zone (SVZ) involvement in glioblastoma. METHODS: We analyzed T1-weighted, gadolinium-enhanced MR images of a retrospective cohort of 647 primary glioblastoma patients diagnosed between 2005-2013, and performed a multivariable Cox regression analysis to adjust the prognostic effect of SVZ involvement for clinical patient- and tumor-related factors. Protein expression patterns of a.o. markers of neural stem cellness (CD133 and GFAP-δ) and (epithelial-) mesenchymal transition (NF-κB, C/EBP-ß and STAT3) were determined with immunohistochemistry on tissue microarrays containing 220 of the tumors. Molecular classification and mRNA expression-based gene set enrichment analyses, miRNA expression and SNP copy number analyses were performed on fresh frozen tissue obtained from 76 tumors. Confirmatory analyses were performed on glioblastoma TCGA/TCIA data. RESULTS: Involvement of the SVZ was a significant adverse prognostic factor in glioblastoma, independent of age, KPS, surgery type and postoperative treatment. Tumor volume and postoperative complications did not explain this prognostic effect. SVZ contact was associated with increased nuclear expression of the (epithelial-) mesenchymal transition markers C/EBP-ß and phospho-STAT3. SVZ contact was not associated with molecular subtype, distinct gene expression patterns, or markers of stem cellness. Our main findings were confirmed in a cohort of 229 TCGA/TCIA glioblastomas. CONCLUSION: In conclusion, involvement of the SVZ is an independent prognostic factor in glioblastoma, and associates with increased expression of key markers of (epithelial-) mesenchymal transformation, but does not correlate with stem cellness, molecular subtype, or specific (mi)RNA expression patterns.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Glioblastoma/genética , Ventrículos Laterais/metabolismo , Fator de Transcrição STAT3/genética , Antígeno AC133/genética , Antígeno AC133/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/cirurgia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Variações do Número de Cópias de DNA , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glioblastoma/diagnóstico por imagem , Glioblastoma/mortalidade , Glioblastoma/cirurgia , Humanos , Ventrículos Laterais/diagnóstico por imagem , Ventrículos Laterais/patologia , Ventrículos Laterais/cirurgia , Imagem por Ressonância Magnética , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Polimorfismo de Nucleotídeo Único , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fator de Transcrição STAT3/metabolismo , Carga Tumoral
9.
BMC Complement Altern Med ; 19(1): 246, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488172

RESUMO

BACKGROUND: Yangkyuksanwha-tang (YST) is an herbal medicine based on Sasang constitutional medicine (SCM) and is widely used in Korean traditional medicine. The aim of the study was to evaluate the effect of YST on obesity in high-fat diet (HFD)-induced obese mice. METHODS: We induced obesity in C57bl/6 J mice using a HFD, and then orally administered 300 mg/kg YST for 6 weeks. We measured body weight, food efficiency, organ and fat weight, serum biochemical parameters, and obesity-related gene expression, and carried out histological analysis at the end of the experimental period. RESULTS: YST significantly reduced the absolute body weight and food efficiency ratio. The serum, aminotransferase, glucose, total cholesterol, triglyceride, and low-density lipoprotein-cholesterol levels were significantly lower in the YST-treated group than in the control group, whereas the high-density lipoprotein-cholesterol level in the YST-treated group was significantly higher. The YST-treated group also showed a significant reduction in regional fatty tissues and the absolute weight of various organs. We also observed a significantly reduced expression of AP2/FABP4, C/EBP-ß, leptin, and SREBP1c/ADD1 mRNA, and significantly increased expression of UCP-2 and adiponectin mRNA in adipose tissue in the YST-treated group. YST also decreased the lipid droplet size and lipid accumulation in the liver, as well as adipocyte size in epididymal adipose tissue. At the dose tested, YST was non-toxic to the liver and kidneys of the mice. CONCLUSION: The results imply that YST has anti-obesity effects in obesity-induced mice. Although the number of experimental animals was limited and the drug effects concern mice, rather than humans, which have different constitutions, the study has valuable implications with respect to the general effects of YST.


Assuntos
Fármacos Antiobesidade/administração & dosagem , Obesidade/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Animais , Fármacos Antiobesidade/química , Peso Corporal/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Humanos , Leptina/genética , Leptina/metabolismo , Masculino , Medicina Tradicional Coreana , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Extratos Vegetais/química , Plantas Medicinais/química , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
10.
Am J Physiol Lung Cell Mol Physiol ; 317(5): L525-L536, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411059

RESUMO

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, ß-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-ß-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-ß, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.


Assuntos
Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperóxia/genética , Oxigênio/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proliferação de Células/efeitos dos fármacos , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Etoposídeo/farmacologia , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feto , Fibroblastos/citologia , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Hiperóxia/metabolismo , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Cultura Primária de Células , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
11.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194412, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31356989

RESUMO

Non-alcoholic steatohepatitis (NASH) is one of the most predominant disorders in metabolic syndrome. Induction of pro-inflammatory mediators in hepatocytes exposed to free fatty acids represents a hallmark event during NASH pathogenesis. C-reactive protein (CRP) is a prototypical pro-inflammatory mediator. In the present study, we investigated the mechanism by which megakaryocytic leukemia 1 (MKL1) mediates palmitate (PA) induced CRP transcription in hepatocytes. We report that over-expression of MKL1, but not MKL2, activated the CRP promoter whereas depletion or inhibition of MKL1 repressed the CRP promoter. MKL1 potentiated the induction of the CRP promoter activity by PA treatment. Importantly, MKL1 knockdown by siRNA or pharmaceutical inhibition by CCG-1423 attenuated the induction of endogenous CRP expression in hepatocytes. Similarly, primary hepatocytes isolated from wild type (WT) mice produced more CRP than those isolated from MKL1 deficient (KO) mice when stimulated with PA. Mechanistically, the sequence-specific transcription factor CCAAT-enhancer-binding protein (C/EBPß) interacted with MKL1 and recruited MKL1 to activate CRP transcription. Reciprocally, MKL1 modulated C/EBPß activity by recruiting the chromatin remodeling protein BRG1 to the CRP promoter to alter histone modifications. In conclusion, our data delineate a novel epigenetic mechanism underlying augmented hepatic inflammation during NASH pathogenesis.


Assuntos
Proteína C-Reativa/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , DNA Helicases/genética , Hepatopatia Gordurosa não Alcoólica/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Animais , Proteína C-Reativa/química , Proteína beta Intensificadora de Ligação a CCAAT/química , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Regiões Promotoras Genéticas , Transativadores/química
12.
Cancer Biother Radiopharm ; 34(8): 537-546, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31314588

RESUMO

Introduction: Growth differentiation factor 15 (GDF15), a newly identified member of transforming growth factor (GDF) superfamily, is upregulated in ovarian (OV) cancer. Upregulated GDF15 positively correlates with poor prognosis of OV cancer. Thus, elucidation of the mechanism underlying GDF15 overexpression is important. Method and Results: PROMO and JASPAR prediction software were used to find transcription factors for GDF15 expression. Data from TCGA database were analyzed to find long noncoding RNAs (lncRNAs) that were also abnormally expressed in OV cancer and had associations with GDF15 expression. Transcription factor CEBPB was predicted as an important regulator of GDF15, confirmed by luciferase reporter assay. However, CEBPB expression was not significantly changed in OV cancer. Data from TCGA database showed that lncRNA GAS5 is downregulated in OV cancer and its expression is negatively correlated with GDF15 expression. RPISeq showed high affinity of GAS5 to CEBPB and this was confirmed by RNA-binding protein immunoprecipitation assay. GAS5 overexpression increased its binding to CEBPB and consequently downregulated GDF15. GAS5 overexpression and GDF15 knockdown decreased viability and increased apoptosis of OV cancer cells, but CEBPB overexpression had opposite effects. However, simultaneous GAS5 and CEBPB overexpression or CEBPB overexpression together with GDF15 knockdown had no effect on cell viability and apoptosis. Conclusion: GAS5 functions as decoy of CEBPB, blocking transcription-promoting effect of CEBPB on GDF15.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Fator 15 de Diferenciação de Crescimento/metabolismo , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Estudos de Casos e Controles , Feminino , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Prognóstico , RNA Longo não Codificante/genética , Células Tumorais Cultivadas
13.
J Biol Chem ; 294(24): 9642-9654, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31061100

RESUMO

ß-Catenin signaling is triggered by WNT proteins and is an important pathway that negatively regulates adipogenesis. However, the mechanisms controlling the expression of WNT proteins during adipogenesis remain incompletely understood. Lysine demethylase 5A (KDM5A) is a histone demethylase that removes trimethyl (me3) marks from lysine 4 of histone 3 (H3K4) and serves as a general transcriptional corepressor. Here, using the murine 3T3-L1 preadipocyte differentiation model and an array of biochemical approaches, including ChIP, immunoprecipitation, RT-qPCR, and immunoblotting assays, we show that Kdm5a is a target gene of CCAAT/enhancer-binding protein ß (C/EBPß), an important early transcription factor required for adipogenesis. We found that C/EBPß binds to the Kdm5a gene promoter and transactivates its expression. We also found that siRNA-mediated KDM5A down-regulation inhibits 3T3-L1 preadipocyte differentiation. The KDM5A knockdown significantly up-regulates the negative regulator of adipogenesis Wnt6, having increased levels of the H3K4me3 mark on its promoter. We further observed that WNT6 knockdown significantly rescues adipogenesis inhibited by the KDM5A knockdown. Moreover, we noted that C/EBPß negatively regulates Wnt6 expression by binding to the Wnt6 gene promoter and repressing Wnt6 transcription. Further experiments indicated that KDM5A interacts with C/EBPß and that their interaction cooperatively inhibits Wnt6 transcription. Of note, C/EBPß knockdown impaired the recruitment of KDM5A to the Wnt6 promoter, which had higher H3K4me3 levels. Our results suggest a mechanism involving C/EBPß and KDM5A activities that down-regulates the Wnt/ß-catenin pathway during 3T3-L1 preadipocyte differentiation.


Assuntos
Adipócitos/citologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Ativação Transcricional , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Camundongos , Regiões Promotoras Genéticas , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína Wnt1/genética , beta Catenina/genética
14.
Br Poult Sci ; 60(4): 347-356, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31064204

RESUMO

1. CCAAT/enhancer binding proteins (C/EBPs), as a family of transcription factors, consists of six functionally and structurally related proteins which share a conserved basic leucine zipper (bZIP) DNA-binding domain. The aim of this study was to clone the full-length coding sequences (CDS) of C/EBP-α and -ß genes, and determine the abundance of these two genes in various tissues of white king pigeon (C. livia). 2. The complete cDNA sequences of C/EBP-α and -ß genes were cloned from pigeons by using PCR combined with rapid amplification of cDNA ends (RACE). The sequences were bioinformatically analysed, and the tissue distribution determined by quantitative real-time RT-PCR (qRT-PCR). 3. The results showed that the full-length cDNA sequences of pigeon C/EBP-α and -ß genes were 2,807bp and 1,778bp, respectively. The open reading frames of C/EBP-α (978 bp) and -ß (987bp) encoded 325 amino acids and 328 amino acids, respectively. The pigeon C/EBP-α and C/EBP-ß proteins were predicted to have a conserved basic leucine zipper (bZIP) domain, which is a common structure feature of the C/EBP family. Multiple sequence alignments indicated that pigeon C/EBP-α and -ß shared more than 90% amino-acid identity with their corresponding homologues in other avian species. Phylogenetic analysis revealed that these two proteins were highly conserved across different species and evolutionary processes. QRT-PCR results indicated that the pigeon C/EBP-α and -ß mRNA transcripts were expressed in all investigated organs. The mRNA expression levels of pigeon C/EBP-α in descending order, were in spleen, heart, liver, lung, kidney and muscle. The pigeon C/EBP-ß gene had the most abundant expression in lung, followed by the kidney, with minimal expression detected in muscle. 4. This study investigated the full-length cDNA sequences, genetic characteristics and tissue distribution of pigeon C/EBP-α and -ß genes and found that they may have functions in various tissues of pigeon. This provides a foundation for further study for regulatory mechanisms of these two genes in birds.


Assuntos
Proteínas Aviárias/genética , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Columbidae/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/química , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Clonagem Molecular , Columbidae/metabolismo , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária
15.
Arch Biochem Biophys ; 671: 235-244, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31071302

RESUMO

Transforming growth factor ß (TGFß) has participated in a variety of cellular biological processes. Smad2 and Smad3 are equally important TGFß downstream effectors in mediating TGFß signals. However, genes involved in controlling the balance between these two signaling pathways are unknown. In this study, we showed that although Smad2 and Smad3 are structurally similar, with 89% amino acid sequence similarity in bovine, Smad3 significantly decreased Smad2 mRNA and protein expression during bovine myoblast differentiation, but not by binding on its promoter. Luciferase assays and electrophoretic mobility shift assays (EMSA) demonstrated that the transcription factors C/EBPα and C/EBPß activate Smad2 promoter activity and expression under high serum medium (GM), whereas the opposite was observed under low serum medium (DM). Moreover, over-expression and interference assays revealed that Smad3 has a different effect on C/EBPα and C/EBPß expression under GM versus DM conditions. After mutation of the C/EBPα and C/EBPß binding sites, Smad3 had a reduced effect on Smad2 promoter activity. Therefore, these results demonstrated that Smad3 inhibits Smad2 expression via its transcription factors C/EBPα and C/EBPß during bovine myoblast differentiation. This novel mechanism of the Smad2/3 genes may offer clues for further investigation of TGFß signal function.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Mioblastos/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Bovinos , Diferenciação Celular/fisiologia , DNA/metabolismo , Masculino , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Smad2/genética , Sítio de Iniciação de Transcrição
16.
Toxicol Lett ; 312: 11-21, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059759

RESUMO

Methamphetamine (METH) is a widely abused illicit psychoactive drug. Our previous study has shown that CCAAT-enhancer binding protein ß (C/EBPß) is an important regulator in METH-induced neuronal autophagy and apoptosis. However, the detailed molecular mechanisms underlying this process remain poorly understood. Previous studies have demonstrated that DNA damage-inducible transcript 4 (DDIT4), Trib3 (tribbles pseudo kinase 3), alpha-synuclein (α-syn) are involved in METH-induced dopaminergic neurotoxicity. We hypothesized that C/EBPß is involved in METH-induced DDIT4-mediated neuronal autophagy and Trib3-mediated neuronal apoptosis. We tested our hypothesis by examining the effects of silencing C/EBPß, DDIT4, Trib3 or α-syn with small interfering ribonucleic acid (siRNA) on METH-induced autophagy and apoptosis in the human neuroblastoma SH-SY5Y cells. We also measured the levels of phosphorylated tuberous sclerosis complex 2 (TSC2) protein and Parkin protein level in SH-SY5Y cells. Furthermore, we demonstrated the effect of silencing C/EBPß on METH-caused neurotoxicity in the striatum of rats by injecting LV-shC/EBPß lentivirus using a stereotaxic positioning system. The results showed that METH exposure increased C/EBPß, DDIT4 protein expression. Elevated DDIT4 expression raised up p-TSC2/TSC2 protein expression ratio, inhibited mTOR signaling pathway, activating cell autophagy. We also found that METH exposure increased the expression of Trib3, α-syn, decreased the Parkin protein expression. Lowering levels of Parkin raised up α-syn expression, which initiated mitochondrial apoptosis by down-regulating anti-apoptotic Bcl-2, followed by up-regulation of pro-apoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. These findings were supported by data showing METH-induced autophagy and apoptosis was significantly inhibited by silencing C/EBPß, DDIT4, Trib3 or α-syn, or by Parkin over-expression. Based on the present data, a novel of mechanism on METH-induced cell toxicity is proposed, METH exposure increased C/EBPß protein expression, triggered DDIT4/TSC2/mTOR signaling pathway, and evoked Trib3/Parkin/α-syn-related mitochondrial apoptotic signaling pathway. Collectively, these results suggest that C/EBPß plays an important role in METH-triggered autophagy and apoptosis and it may be a potential target for therapeutics in METH-caused neurotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Estimulantes do Sistema Nervoso Central/toxicidade , Metanfetamina/toxicidade , Neurônios/efeitos dos fármacos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Masculino , Neuroblastoma , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
17.
Neoplasia ; 21(6): 545-556, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31042624

RESUMO

CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPß) was upregulated by metformin and that two isoforms of CEBPß reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPß signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.


Assuntos
Antígeno AC133/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Quinases/genética , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Metformina/farmacologia , Camundongos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Mol Immunol ; 112: 72-81, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31078118

RESUMO

Myeloid-derived suppressor cells (MDSC) expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBPß synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBPß protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPß protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.


Assuntos
Calgranulina B/genética , MicroRNAs/genética , Células Mieloides/metabolismo , Células Supressoras Mieloides/metabolismo , Sepse/genética , Animais , Antígenos Ly/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Antígeno CD11b/genética , Modelos Animais de Doenças , Imunossupressão/métodos , Inflamação/genética , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética
19.
Am J Physiol Endocrinol Metab ; 316(6): E1081-E1092, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30964708

RESUMO

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPß gene. Notably, C/EBPß also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPß and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPß alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPß binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPß. C/EBPß binds directly to the promoter of the musclin gene and upregulates its expression.


Assuntos
Fator 4 Ativador da Transcrição/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Palmitatos/farmacologia , Fatores de Transcrição/efeitos dos fármacos , eIF-2 Quinase/efeitos dos fármacos , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , eIF-2 Quinase/metabolismo
20.
Cancer Sci ; 110(6): 2050-2062, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945396

RESUMO

The PPAR coactivator-1α (PGC1α) is an important transcriptional co-activator in control of fatty acid metabolism. Mitochondrial fatty acid oxidation (FAO) is the primary pathway for the degradation of fatty acids and promotes NADPH and ATP production. Our previous study demonstrated that upregulation of carnitine palmitoyl transferase 1 A (CPT1A), the key regulator of FAO, promotes radiation resistance of nasopharyngeal carcinoma (NPC). In this study, we found that high expression of PGC1α is associated with poor overall survival in NPC patients after radiation treatment. Targeting PGC1α could sensitize NPC cells to radiotherapy. Mechanically, PGC1α binds to CCAAT/enhancer binding protein ß (CEBPB), a member of the transcription factor family of CEBP, to promote CPT1A transcription, resulting in activation of FAO. Our results revealed that the PGC1α/CEBPB/CPT1A/FAO signaling axis promotes radiation resistance of NPC. These findings indicate that the expression of PGC1α could be a prognostic indicator of NPC, and targeting FAO in NPC with high expression of PGC1α might improve the therapeutic efficacy of radiotherapy.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Carnitina O-Palmitoiltransferase/genética , Ácidos Graxos/metabolismo , Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/radioterapia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Estimativa de Kaplan-Meier , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Oxirredução/efeitos da radiação , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos da radiação , Interferência de RNA
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