Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.390
Filtrar
1.
Mol Cell Biol ; 40(17)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601106

RESUMO

Transcription factors C/EBPß and C/EBPδ are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα and are required for adipogenesis in vivo However, the mechanism that controls the induction of C/EBPß and C/EBPδ remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα during adipogenesis. Here, we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated glucocorticoid receptor to directly control the induction of C/EBPß and C/EBPδ in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBPß and C/EBPδ and leads to severe defects in adipogenesis. Adipogenesis defects in PAGR1-deficient cells can be rescued by the ectopic expression of C/EBPß or PPARγ. Finally, the deletion of Pagr1 in Myf5+ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBPß and C/EBPδ, PAGR1 plays a critical role in adipogenesis.


Assuntos
Adipogenia/fisiologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Diferenciação Celular/fisiologia , Histona Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , PPAR gama/metabolismo , Ligação Proteica
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.
Invest Ophthalmol Vis Sci ; 61(3): 39, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32196098

RESUMO

Purpose: Inflammation, hyaluronan production, and adipogenesis are the main pathological events leading to Graves' orbitopathy (GO). Guggulsterone (GS), a phytosterol found in the resin of the guggul plant, is a well-known treatment for several inflammatory disorders, such as arthritis, obesity, and hyperlipidemia. Here we investigated the effects of GS treatment on GO pathology. Methods: Using primary cultures of orbital fibroblasts from GO patients and non-GO controls, we examined the effects of GS on hyaluronan production and the production of proinflammatory cytokines induced by interleukin (IL)-1ß, using real-time reverse transcription-polymerase chain reaction analysis, western blots, and enzyme-linked immunosorbent assays. Further, adipogenic differentiation was evaluated by quantification of Oil Red O staining and assessment of protein levels of peroxisome proliferator activator gamma (PPARγ), CCAAT-enhancer-binding proteins (C/EBP) α and ß, and sterol regulatory element-binding protein-1 (SREBP-1). Results: Treatment with noncytotoxic concentrations of GS resulted in the dose-dependent inhibition of IL-1ß-induced inflammatory cytokines, including IL-6, IL-8, MCP-1, and COX-2, at both mRNA and protein levels. The hyaluronan level was also significantly suppressed by GS. Moreover, GS significantly decreased the formation of lipid droplets and expression of PPARγ, C/EBP α/ß, and SREBP-1 in a dose-dependent manner. GS pretreatment attenuated the phosphorylation of nuclear factor-kappa B induced by IL-1ß. Conclusions: Our data show significant inhibitory effects of GS on inflammation, production of hyaluronan, and adipogenesis in orbital fibroblasts. To our knowledge, this is the first in vitro preclinical evidence of the therapeutic effect of GS in GO.


Assuntos
Fibroblastos/efeitos dos fármacos , Oftalmopatia de Graves/tratamento farmacológico , Órbita/efeitos dos fármacos , Pregnenodionas/uso terapêutico , Adipogenia/efeitos dos fármacos , Adulto , Idoso , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Células Cultivadas , Commiphora/química , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/metabolismo , Oftalmopatia de Graves/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Masculino , Pessoa de Meia-Idade , Órbita/metabolismo , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Adulto Jovem
4.
Biol Pharm Bull ; 43(3): 503-508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115509

RESUMO

Obesity elevates the risk of cardiovascular disease and has been strongly associated with increases in the incidence of many metabolic diseases. Therefore, prevention of obesity leads to the prevention of metabolic diseases. In light of this, substances that exert anti-obesity effects are crucial for the prevention of obesity. Indirubin, a 3,2' bisindole isomer of indigo, is the active component of the traditional Chinese medicine used for the treatment of chronic myelocytic leukemia. In particular, indirubin-3'-oxime (1) was shown to inhibit the differentiation of adipocytes. In this study, we investigated the inhibitory effects of nine indirubin-3'-oxime derivatives against lipid accumulation during differentiation in 3T3-L1 cells. Among the compounds tested, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) at 5 µM exhibited significantly stronger inhibitory activity than indirubin-3'-oxime (1). Furthermore, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) markedly suppressed the expression of CCAAT/enhancer-binding protein α, peroxisome proliferator activator γ2, and adipocyte protein 2, both of which are key adipogenic regulators at the intermediate stage of adipocyte differentiation. Our results demonstrate that 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) significantly down-regulated lipid accumulation during differentiation of 3T3-L1 cells, suggesting their potential as novel therapeutic drugs against the development of obesity.


Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Indóis/química , Indóis/farmacologia , Camundongos , Oximas , Extratos Vegetais/farmacologia
5.
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
6.
Nucleic Acids Res ; 48(7): 3513-3524, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32095812

RESUMO

The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanisms controlling its transcription are likely divergent between them. Here, we determine how two extragenic CREs that are prominent in epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44 and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here, we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter, suggesting non-redundant enhancers. The deletions also greatly reduced promoter interactions with the 5' TAD boundary. We show substantial recruitment of RNAPII to the -35 kb element and identify CEBPß as a key activator of airway expression of CFTR, likely through occupancy at this CRE and the gene promoter.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Elementos Facilitadores Genéticos , Mucosa Respiratória/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Sistemas CRISPR-Cas , Células CACO-2 , Linhagem Celular , Cromatina/química , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Células Epiteliais/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Deleção de Sequência , Transativadores/metabolismo
7.
Biochim Biophys Acta Gene Regul Mech ; 1863(2): 194488, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31926341

RESUMO

Polo-like kinase 4 (PLK4) is a member of the serine/threonine protein kinase family involved in cell-cycle regulation and cellular response to stresses. However, the alteration of PLK4 in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we focused on the regulation of PLK4 regulation in response to ER stress. PLK4 expression was dramatically reduced under ER stress induced by brefeldin A (BFA), tunicamycin (TM), or thapsigargin (TG) and down regulation of PLK4 expression was dependent on activating transcription factor 6 (ATF6) and CCAAT/enhancer-binding protein ß (C/EBPß). Luciferase activity analysis of the truncated PLK4 promoter indicated that region from -1343 to -1250 of the PLK4 promoter was sensitive to BFA or TG. Additionally, ChIP and ChIP Re-IP assays showed that ATF6 and C/EBPß were assembled on the same region of Plk4 promoter. Notably, we identified one C/EBPß responsive element at position -1284, to which ATF6 or C/EBPß binding was enhanced by BFA or TG under in vitro and in vivo conditions. Finally, overexpression of PLK4 inhibits apoptosis and promotes cell proliferation in response to ER stress. In summary, these results demonstrated that ER stress plays a crucial role in PLK4 expression. ATF6 may upregulate DNA-binding affinities after BFA treatment, via recruiting C/EBPß to the upstream promoter of PLK4. These findings may contribute to the understanding of the molecular mechanism of PLK4 regulation.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Neoplasias Ósseas/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/genética , Proteínas Serina-Treonina Quinases/genética , Apoptose , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mutagênese , Osteossarcoma/enzimologia , Osteossarcoma/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Elementos de Resposta , Transcrição Genética
8.
Med Sci Monit ; 26: e918599, 2020 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-31955176

RESUMO

BACKGROUND The aim of this study was to explore the influence of mitofusin-2 (Mfn-2) on phosphatidylinositol transfer protein 3 (PITPNM3) and tumor growth and the potential mechanism behind the regulation of Mfn-2 on PITPNM3 in hepatic carcinoma cell line SMMC-7721. MATERIAL AND METHODS We obtained promoter sequence of PITPNM3 gene from University of Santa Cruz (UCSC) genomic database, and we predict transcriptional factor of PITPNM3 genes by JASPAR database. Target transcription factor was determined by comparison of binding sites number for promoter. SMMC-7721 cells were transfected with expression plasmid containing Mfn-2, transcription factor gene and PITPNM3. The cells transfected with empty vector were used as control. Real-time polymerase chain reaction was used to determine the mRNA level of target genes. Co-immunoprecipitation (Co-IP) assay was used to determine the interaction between Mfn-2 and target transcription factor. Chromatin immunoprecipitation assay (ChIP) assay was used to determine the binding of transcription factor with PITPNM3 promoter. Tumorigenicity assay was used to compare the effect of Mfn-2, SP1, and PITPNM3 on tumor development. RESULTS SP1 was selected as the target transcriptional factor. In the Co-IP assay, Mfn-2 was shown to interact with SP1. In the ChIP assay Mfn-2 transfection resulted in decreased binding number of SP1 with PITPNM3 promoter. Furthermore, PITPNM3 mRNA levels were significantly increased in SMMC-7721 cells transfected with SP1 but were decreased after transfection with Mfn-2. In nude mice, PITPNM3 and SP1 upregulation lead to larger tumor lump and conversely Mfn-2 upregulation lead to smaller tumor lump. CONCLUSIONS Mfn-2 could suppress expression of PITPNM3 through interaction with transcription factor SP1; Mfn-2 may have anti-tumor activity; SP1 and PITPNM3 may promote tumor development.


Assuntos
Proteínas de Ligação ao Cálcio/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/genética , Proteínas Mitocondriais/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos Nus , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Regulação para Cima/genética
9.
J Biol Chem ; 295(9): 2787-2803, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31953319

RESUMO

Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in skeletal muscle fibers. However, the direct biochemical mechanism by which ATF4 promotes muscle atrophy is unknown. ATF4 is a member of the basic leucine zipper transcription factor (bZIP) superfamily. Because bZIP transcription factors are obligate dimers, and because ATF4 is unable to form highly-stable homodimers, we hypothesized that ATF4 may promote muscle atrophy by forming a heterodimer with another bZIP family member. To test this hypothesis, we biochemically isolated skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo Interestingly, we found that ATF4 forms at least five distinct heterodimeric bZIP transcription factors in skeletal muscle fibers. Furthermore, one of these heterodimers, composed of ATF4 and CCAAT enhancer-binding protein ß (C/EBPß), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4-C/EBPß heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF-C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBPß, and the ATF-C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemical mechanism by which ATF4 induces skeletal muscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Atrofia Muscular/etiologia , Multimerização Proteica , Fatores Ativadores da Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Camundongos , Músculo Esquelético/patologia
10.
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
11.
Dokl Biol Sci ; 488(1): 133-135, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31732897

RESUMO

C/EBP-ß, a basic leucine zipper transcription factor, has important roles in the regulation of the body immune and inflammatory responses. Wistar rats subjected to combined irradiation were characterized by an increase in the content of the C/EBP-ß LIP isoform in the pituitary gland. The obtained data indicate that moderate doses of ionizing radiation to initiate the endoplasmic reticulum stress response and are likely to initiate C/EBP-ß-mediated cell death according to the apoptotic scenario. This study also confirms the earlier hypothesis about the alterations of the hypothalamic-pituitary-adrenocortical axis in response to moderate doses of ionizing radiation.


Assuntos
Apoptose/efeitos da radiação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Radioisótopos de Carbono , Estresse do Retículo Endoplasmático/efeitos da radiação , Raios gama/efeitos adversos , Hipófise/metabolismo , Animais , Hipófise/patologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar
12.
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
13.
Genes (Basel) ; 10(10)2019 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614865

RESUMO

This study identified a transcription factor that might bind to the 5' regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the -1219 to -1209 bp (ATG+1) region of the HTR1A. Two sequences located in the -722 to -372 bp and -119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence -1219 - -1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.


Assuntos
Região 5'-Flanqueadora , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Receptor 5-HT1A de Serotonina/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Receptor 5-HT1A de Serotonina/metabolismo , Sequências Reguladoras de Ácido Nucleico
14.
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
15.
Immunity ; 51(3): 522-534.e7, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31471107

RESUMO

Although recent progress provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), rare anti-PF therapeutics show definitive promise for treating this disease. Repeated lung epithelial injury results in injury-repairing response and inflammation, which drive the development of PF. Here, we report that chronic lung injury inactivated the ubiquitin-editing enzyme A20, causing progressive accumulation of the transcription factor C/EBPß in alveolar macrophages (AMs) from PF patients and mice, which upregulated a number of immunosuppressive and profibrotic factors promoting PF development. In response to chronic lung injury, elevated glycogen synthase kinase-3ß (GSK-3ß) interacted with and phosphorylated A20 to suppress C/EBPß degradation. Ectopic expression of A20 or pharmacological restoration of A20 activity by disturbing the A20-GSK-3ß interaction accelerated C/EBPß degradation and showed potent therapeutic efficacy against experimental PF. Our study indicates that a regulatory mechanism of the GSK-3ß-A20-C/EBPß axis in AMs may be a potential target for treating PF and fibroproliferative lung diseases.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Macrófagos/metabolismo , Fibrose Pulmonar/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Regulação para Cima/fisiologia
16.
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
17.
Mol Cell Biochem ; 462(1-2): 51-59, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31428903

RESUMO

Interferon-stimulated gene 15 (ISG15) is a member of the family of ubiquitin-like proteins. Similar to ubiquitin, conjugation of ISG15 to cellular proteins requires cascade reactions catalyzed by at least 2 enzymes, UbE1L and UbcH8. Expression of ISG15 and its conjugates is up-regulated in many cancer cells, yet the underlying mechanism of up-regulation is still unclear. In this study, we showed that TNF-α, similar to the response by IFN-ß, could directly induce expression of ISG15 and its conjugation machinery, UbE1L and UbcH8, in human lung carcinoma, A549. The early response of their expression was effectively blocked by specific inhibitors of p38 MAPK (SB202190) and JNK (SP600125), but not by B18R, a soluble type-I IFN receptor. In addition, luciferase reporter assay together with serial deletions and site-directed mutagenesis identified a putative C/EBPß binding element in the ISG15 promoter, which is necessary to the response by TNF-α. Taken together, expression of ISG15 and ISG15 conjugation machinery in cancer cells is directly up-regulated by TNF-α via p38 MAPK and JNK pathways through the activation of C/EBPß binding element in the ISG15 promoter. This study provides a new insight toward understanding the molecular mechanism of ISG15 system and inflammatory response in cancer progression.


Assuntos
Citocinas/genética , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , Fator de Necrose Tumoral alfa/farmacologia , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/metabolismo
18.
Mol Cells ; 42(7): 530-545, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31362469

RESUMO

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24- cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.


Assuntos
Carcinoma Hepatocelular/genética , Quimiocina CXCL12/genética , Histonas/metabolismo , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Microambiente Tumoral/genética , Regulação para Cima/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiocina CXCL12/metabolismo , Epigênese Genética/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Compostos Heterocíclicos/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacologia , Transcrição Genética/efeitos da radiação , Microambiente Tumoral/efeitos da radiação , Raios X
19.
J Clin Lab Anal ; 33(9): e22985, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31364785

RESUMO

OBJECTIVE: To screen the gene related to osteosarcoma (OS) metastasis and the molecular mechanism. METHODS: GEO database and R2 chip analysis platform were used to screen genes related to OS metastasis. UCSC gene browser was used to find the transcription factor (TF) of CLEC5A. The mRNA level and protein expression of CLEC5A in OS tissues and normal tissues were determined by RT-PCR and Western blotting, respectively. OS cell lines MG-63 were transfected with CEBPB recombinant plasmid. After transfection, the expression of CLEC5A in MG-63 cells was determined and the cell proliferation situation was determined by clone formation assay. RESULTS: Three genes CLEC5A, ALOX5AP, and RNASE3 were obtained, and CLEC5A has the highest correlation with OS. CLEC5A has screened the gene related to OS metastasis, and CEBPB can be taken as TF regulating downstream gene CLEC5A. CEBPB can regulate the downstream CLEC5A as transcription factor. The relative mRNA level and protein expression of CLEC5A in OS tissues were significantly higher than those in normal tissues. CLEC5A can prevent OS metastasis. Transfection of CEBPB increased the expression of CLEC5A in MG-63 cells and also inhibited the proliferation of OS. CONCLUSION: CEBPB can inhibit the proliferation of OS cells via regulating the expression of CLEC5A.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação Neoplásica da Expressão Gênica , Lectinas Tipo C/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Receptores de Superfície Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Clonais , Regulação para Baixo/genética , Testes Genéticos , Humanos , Lectinas Tipo C/metabolismo , Metástase Neoplásica , Receptores de Superfície Celular/metabolismo
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA