RESUMO
MYO18B has been proposed to contribute to the progression of hepatocellular carcinoma (HCC). However, the signals that govern MYO18B transcription are not known. Here we show that, a network of C19MC miRNA-520G, IFN-γ, CEBPB and p53 transcriptional-defects promote MYO18B mRNA expression in HCCs. IFN-γ by itself suppresses MYO18B transcription, but promotes it when miRNA-520G is stably overexpressed. Similarly, CEBPB-liver-enriched activator protein (LAP) isoform overexpression suppresses MYO18B transcription but promotes transcription when the cells are treated with IFN-γ. Furthermore, miR-520G together with mutant-p53 promotes MYO18B transcription. Conversely, bFGF suppresses MYO18B mRNA irrespective of CEBPB, miR-520G overexpression or IFN-γ treatment. Finally high MYO18B expression reflects poor prognosis while high MYL5 or MYO1B expression reflects better survival of HCC patients. Thus, we identified a network of positive and negative regulators of MYO18B mRNA expression which reflects the survival of HCC patients.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Carcinoma Hepatocelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Regulação Neoplásica da Expressão Gênica , Interferon gama/biossíntese , Neoplasias Hepáticas/metabolismo , MicroRNAs/biossíntese , Miosinas/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Interferon gama/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/genética , Miosinas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Introduction of targeted therapy in the treatment of metastatic cutaneous malignant melanoma (CMM) has improved clinical outcome during the last years. However, only in a subset of the CMM patients, this will lead to long-term effects. CEBPB is a transcription factor that has been implicated in various physiological and pathological processes, including cancer development. We have investigated its prognostic impact on CMM and unexpectedly found that higher CEBPB mRNA levels correlated with a longer overall survival. Furthermore, in a small cohort of patients with metastatic CMM treated with BRAF-inhibitors, higher levels of CEBPB mRNA expression in the tumor cells prior treatment correlated to a longer progression-free survival. We have characterized an overlapping antisense transcript, CEBPB-AS1, with the aim to investigate the regulation of CEBPB expression in CMM and its impact on BRAF-inhibitor sensitivity. We demonstrated that silencing of CEBPB-AS1 resulted in epigenetic modifications in the CEBPB promoter and in increased CEBPB mRNA and protein levels, inhibited proliferation and partially resensitized BRAF-inhibitor resistant CMM cells to this drug-induced apoptosis. Our data suggest that targeting CEBPB-AS1 may represent a valuable tool to sensitize CMM cells to the BRAF-inhibitor-based therapies.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Melanoma/tratamento farmacológico , RNA Antissenso/genética , Vemurafenib/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , RNA Antissenso/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPß, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/EBPδ was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARγ antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50⯵M DINP. The GO enrichment showed the GO term of "fat cell differentiation" was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism.
Assuntos
Adipócitos/citologia , Adipogenia/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Células 3T3-L1 , Tecido Adiposo/metabolismo , Anilidas/farmacologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Linhagem Celular , Regulação para Baixo , Expressão Gênica/efeitos dos fármacos , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Ativação Transcricional , Regulação para CimaRESUMO
BACKGROUND: To investigate the role of microRNA (miR)-27a and miR-27b in adipogenesis in an in vitro model of Graves' orbitopathy (GO). METHODS: Orbital fat tissues were harvested from GO and non-GO participants for primary orbital fibroblast cultures. The expression levels of miR-27a and miR-27b between GO and non-GO orbital fat tissues were compared. During adipogenesis of GO orbital fibroblasts, the expression levels of miR-27a and miR-27b were determined, and the effects of mimics of miR-27a and miR-27b transfection on adipogenesis of GO orbital fibroblast were investigated. RESULTS: Real time-polymerase chain reaction showed significantly more decreases in miR-27a and miR-27b levels in orbital fat tissues in GO participants than in non-GO participants (p < 0.05). The expression of both miR-27a and miR-27b was highest in orbital fibroblasts at day 0 and declined gradually after the induction of adipogenic differentiation. The expression levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and C/EBPß were decreased and Oil Red O-stained lipid droplets were lower in GO orbital fibroblasts transfected with miR-27a and miR-27b mimics than in negative controls. CONCLUSIONS: Our results indicated that miR-27a and miR-27b inhibited adipogenesis in orbital fibroblasts from GO patients. Further studies are required to examine the potential of miR-27a and miR-27b as targets for therapeutic strategies.
Assuntos
Adipogenia , Diferenciação Celular , Fibroblastos/metabolismo , Oftalmopatia de Graves/metabolismo , MicroRNAs/biossíntese , Órbita/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Feminino , Fibroblastos/patologia , Regulação da Expressão Gênica , Oftalmopatia de Graves/patologia , Humanos , Masculino , Órbita/patologia , PPAR gama/biossínteseRESUMO
BACKGROUND: Implantation of rat prostate cancer cells into the normal rat prostate results in tumor-stimulating adaptations in the tumor-bearing organ. Similar changes are seen in prostate cancer patients and they are related to outcome. One gene previously found to be upregulated in the non-malignant part of tumor-bearing prostate lobe in rats was the transcription factor CCAAT/enhancer-binding protein-ß (C/EBPß). METHODS: To explore this further, we examined C/EBPß expression by quantitative RT-PCR, immunohistochemistry, and Western blot in normal rat prostate tissue surrounding slow-growing non-metastatic Dunning G, rapidly growing poorly metastatic (AT-1), and rapidly growing highly metastatic (MatLyLu) rat prostate tumors-and also by immunohistochemistry in a tissue microarray (TMA) from prostate cancer patients managed by watchful waiting. RESULTS: In rats, C/EBPß mRNA expression was upregulated in the surrounding tumor-bearing prostate lobe. In tumors and in the surrounding non-malignant prostate tissue, C/EBPß was detected by immunohistochemistry in some epithelial cells and in infiltrating macrophages. The magnitude of glandular epithelial C/EBPß expression in the tumor-bearing prostates was associated with tumor size, distance to the tumor, and metastatic capacity. In prostate cancer patients, high expression of C/EBPß in glandular epithelial cells in the surrounding tumor-bearing tissue was associated with accumulation of M1 macrophages (iNOS+) and favorable outcome. High expression of C/EBPß in tumor epithelial cells was associated with high Gleason score, high tumor cell proliferation, metastases, and poor outcome. CONCLUSIONS: This study suggest that the expression of C/EBP-beta, a transcription factor mediating multiple biological effects, is differentially expressed both in the benign parts of the tumor-bearing prostate and in prostate tumors, and that alterations in this may be related to patient outcome.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Processos de Crescimento Celular/fisiologia , Estudos de Coortes , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Metástase Neoplásica , Transplante de Neoplasias , Fosforilação , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Análise Serial de TecidosRESUMO
Ageing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBPß-LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the Cebpb-mRNA. Here, we describe that reduced C/EBPß-LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice. Moreover, female C/EBPßΔuORF mice display an extended lifespan. Since LIP levels increase upon aging in wild type mice, our data reveal an important role for C/EBPß in the aging process and suggest that restriction of LIP expression sustains health and fitness. Thus, therapeutic strategies targeting C/EBPß-LIP may offer new possibilities to treat age-related diseases and to prolong healthspan.
Assuntos
Envelhecimento , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Expressão Gênica , Animais , Regulação para Baixo , Feminino , Longevidade , Masculino , Camundongos Endogâmicos C57BLRESUMO
Rosiglitazone (RG) is a well-known activator of peroxisome proliferator-activated receptor-gamma (PPARγ) and used to treat hyperglycemia and type 2 diabetes; however, its clinical application has been confounded by adverse side effects. Here, we assessed the roles of chlorogenic acid (CGA), a phenolic secondary metabolite found in many fruits and vegetables, on the differentiation and lipolysis of mouse 3T3-L1 preadipocytes. The results showed that CGA promoted differentiation in vitro according to oil red O staining and quantitative polymerase chain reaction assays. As a potential molecular mechanism, CGA downregulated mRNA levels of the adipocyte differentiation-inhibitor gene Pref1 and upregulated those of major adipogenic transcriptional factors (Cebpb and Srebp1). Additionally, CGA upregulated the expression of the differentiation-related transcriptional factor PPARγ2 at both the mRNA and protein levels. However, following CGA intervention, the accumulation of intracellular triacylglycerides following preadipocyte differentiation was significantly lower than that in the RG group. Consistent with this, our data indicated that CGA treatment significantly upregulated the expression of lipogenic pathway-related genes Plin and Srebp1 during the differentiation stage, although the influence of CGA was weaker than that of RG. Notably, CGA upregulated the expression of the lipolysis-related gene Hsl, whereas it did not increase the expression of the lipid synthesis-related gene Dgat1. These results demonstrated that CGA might function as a potential PPARγ agonist similar to RG; however, the impact of CGA on lipolysis in 3T3-L1 preadipocytes differed from that of RG.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ácido Clorogênico/farmacologia , Lipólise/efeitos dos fármacos , PPAR gama/agonistas , Células 3T3-L1 , Adipócitos/citologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , PPAR gama/metabolismo , Perilipina-1/biossíntese , Rosiglitazona/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Triglicerídeos/metabolismoRESUMO
Amphibole asbestos exposure is associated with the production of mesothelial cell autoantibodies (MCAA). These MCAA have been linked with pleural fibrotic disease in the asbestos exposed community of Libby, Montana, and induce collagen deposition by cultured mesothelial cells. However, the exact intracellular mechanism by which these autoantibodies cause an increase in collagen deposition remains unknown. This study sought to gain insight into the transcription factors involved in the collagen production after human mesothelial cells are exposed to MCAA. In this study, transcription factor activation profiles were generated from human mesothelial cells (Met5A) treated with serum from Libby subjects, and were compared to cells treated with serum cleared of IgG, and therefore containing no MCAA. Analysis of those profiles indicated C/EBP-beta and hypoxia inducible factor 1 alpha (HIF-1α) are significantly increased in the nucleus, indicating activation, due to MCAA exposure compared to controls. Inhibition of either of these transcription factors significantly reduced collagen 1 deposition by these cells following exposure to MCAA. These data suggest autoantibodies are directly involved in type I collagen deposition and may elucidate potential therapeutic targets for autoantibody mediated fibrosis.
Assuntos
Autoanticorpos/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Células Epiteliais/imunologia , Fibrose/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Amiantos Anfibólicos , Células Cultivadas , Colágeno/metabolismo , Expressão Gênica , Humanos , Exposição Ocupacional , Soro , Regulação para CimaRESUMO
Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPß and EBF2, suggesting their functional cooperation during adipogenesis.
Assuntos
Adipogenia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Homeodomínio/genética , Obesidade/genética , Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Marrom/metabolismo , Animais , Autoantígenos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais , Camundongos , Obesidade/metabolismo , Obesidade/patologia , RNA Longo não Codificante/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
The differentiation of preadipocytes into adipocytes is controlled by several transcription factors, including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), which are known as master regulators of adipogenesis. BCL11B is a zinc finger-type transcription factor that regulates the development of the skin and central nervous and immune systems. Here, we found that BCL11B was expressed in the white adipose tissue (WAT), particularly the subcutaneous WAT and that BCL11B(-/-) mice had a reduced amount of subcutaneous WAT. During adipogenesis, BCL11B expression transiently increased in 3T3-L1 preadipocytes and mouse embryonic fibroblasts (MEFs). The ability for adipogenesis was reduced in BCL11B knockdown 3T3-L1 cells and BCL11B(-/-) MEFs, whereas the ability for osteoblastogenesis was unaffected in BCL11B(-/-) MEFs. Luciferase reporter gene assays revealed that BCL11B stimulated C/EBPß activity. Furthermore, the expression of downstream genes of the Wnt/ß-catenin signaling pathway was not suppressed in BCL11B(-/-) MEFs during adipogenesis. Thus, this study identifies BCL11B as a novel regulator of adipogenesis, which works, at least in part, by stimulating C/EBPß activity and suppressing the Wnt/ß-catenin signaling pathway.
Assuntos
Adipogenia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteínas Repressoras/metabolismo , Gordura Subcutânea/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Adipócitos/fisiologia , Animais , Células Cultivadas , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Proteínas Repressoras/deficiência , Proteínas Supressoras de Tumor/deficiência , Via de Sinalização WntRESUMO
Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia (AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinase C (PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRAinduced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogenactivated protein kinase kinase (MEK) and extracellular signalregulated kinase (ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancerbinding protein ß (C/EBPß) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporineenhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRAinduced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Leucemia Mieloide Aguda/tratamento farmacológico , Estaurosporina/administração & dosagem , Tretinoína/administração & dosagem , Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/biossíntese , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Inibidores de Proteínas Quinases/administração & dosagem , Células U937RESUMO
Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC). Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF). Bacterial enterotoxin activates spermine oxidase (SMO), which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPß (CREBP), and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPß may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT) as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPß rather than ETBF infection.
Assuntos
Infecções por Bacteroides/patologia , Bacteroides fragilis/isolamento & purificação , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/microbiologia , Poliaminas/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Infecções por Bacteroides/genética , Infecções por Bacteroides/metabolismo , Infecções por Bacteroides/microbiologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Expressão Gênica , Genes myc , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Proteínas Proto-Oncogênicas c-myc/genética , Poliamina OxidaseRESUMO
MEK/ERK signal pathway was required for the differentiation of granulocytes, megakaryocytes and erythrocytes. Recently, MEK/ERK cascade was reported to be involved in all-trans retinoic acid (ATRA) induced differentiation in acute promyelocytic leukemia (APL) cells. However, the upstream and downstream molecules of MEK/ERK signal pathway in this cell model remains to be elucidated. In this work, we showed that RAF-1 was activated and the blockade of RAF-1 activation attenuated MEK/ERK activation as well as ATRA-induced differentiation. ATRA-enhanced protein levels of C/EBPß, C/EBPε and PU.1, which were required for differentiation in APL cells, were suppressed by the specific inhibitor of MEK. However, MEK inhibition had no effect on the degradation of PML-RARα fusion protein or the restoration of PML nuclear bodies by ATRA treatment. Taken together, our study suggested that RAF-1/MEK/ERK cascade was involved in ATRA-induced differentiation in APL cells through enhancing the protein level of C/EBPß, C/EBPε and PU.1.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Linhagem Celular Tumoral , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Transativadores/biossíntese , Transativadores/efeitos dos fármacosRESUMO
Persistent exposure of rats to 6-propyl-2-thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein ß (C/EBP-ß). Catalase promoter region (-185 to +52) that contains binding sites for C/EBP-ß showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP-ß to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid-disrupting properties of PTU. It is possible that besides thyroid-disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Catalase/biossíntese , Propiltiouracila/administração & dosagem , Glândula Tireoide/efeitos dos fármacos , Hormônios Tireóideos/biossíntese , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Catalase/genética , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Regiões Promotoras Genéticas , Ratos , Glândula Tireoide/patologia , Hormônios Tireóideos/genéticaRESUMO
AIM: Pregnancy is a physiological model of adaptive and reversible heart enlargement, but the molecular mechanisms determining this kind of physiologic cardiac hypertrophy are poorly known. Here, we analyzed the role of the transcription factor C/EBPß in the development of pregnancy-induced cardiac hypertrophy. RESULTS: C/EBPß+/- mice at day 18 of gestation were used as happloinsufficiency model of late pregnancy. We found that C/EBPß expression was specifically increased in hearts from Wt pregnant mice whereas expression of other C/EBP subtypes (α and δ) was not affected by gestation. Pregnancy-induced changes in systemic metabolic and hormonal profiles were not essentially different in Wt versus C/EBPß+/- mice. However, C/EBPß+/- mice developed pregnancy-induced heart hypertrophy to a lower extent relative to Wt mice. Furthermore, hearts from C/EBPß+/- mice have alterations in fatty acid oxidation genes and reductions in the expression levels of glucose transporters that may compromise metabolic cardiac function during pregnancy. Among marker genes of inflammation, interleukin-6 (Il-6) showed a marked differential behavior in C/EBPß+/- pregnant mice: pregnancy strongly induced cardiac Il-6 expression in wt, a phenomenon that did not occur in C/EBPß+/- mice. Moreover, marker genes for M2 macrophages were decreased in C/EBPß+/- pregnant mice and in C/EBPß-/- mice subjected to LPS stimulus. CONCLUSIONS: Here we found that normal levels of C/EBPß are required for hypertrophy development during pregnancy. Events such as the increase in IL-6 in the heart of pregnant mice are prevented in C/EBPß+/- animals. Moreover, C/EBPß controls M2-macrophage gene expression in the heart. Thus, C/EBPß appears as a transcription factor required for cardiac hypertrophy response to gestation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Cardiomegalia/genética , Regulação da Expressão Gênica no Desenvolvimento , Prenhez , RNA/genética , Animais , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Cardiomegalia/diagnóstico , Cardiomegalia/metabolismo , Ecocardiografia , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Gravidez , Complicações Cardiovasculares na GravidezRESUMO
Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4+FoxP3- T cells expressing Helios (FoxP3-Helios+) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4+Helios+ T cells regardless of FoxP3 expression (FoxP3+/-Helios+). We show that CD4+GARP+/-LAP+ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3+Helios+ Tregs proliferate in vitro significantly less than FoxP3+Helios- Tregs upon TCR stimulation. Unlike FoxP3+Helios- Tregs, FoxP3+Helios+ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3+Helios+ Tregs have more suppressive characteristics, compared with FoxP3+Helios- Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3+Helios+ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Fatores de Transcrição Forkhead/imunologia , Fator de Transcrição Ikaros/imunologia , Proteínas de Membrana/imunologia , Linfócitos T Reguladores/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/sangue , Células Cultivadas , Fatores de Transcrição Forkhead/sangue , Humanos , Fator de Transcrição Ikaros/sangue , Interleucina-10/biossíntese , Interleucina-10/sangue , Interleucina-10/imunologia , Ativação Linfocitária , Proteínas de Membrana/biossíntese , Proteínas de Membrana/sangueRESUMO
Chemoresistance to anticancer drugs substantially reduces survival in epithelial ovarian carcinoma (EOC). Here, microarray analysis showed that collagen type XI alpha 1 (COL11A1) is a chemotherapy response-associated gene. Chemoresistant cells expressed higher COL11A1 and c/EBPß than chemosensitive cells. COL11A1 or c/EBPß downregulation suppressed chemoresistance, whereas COL11A1 overexpression attenuated sensitivity to cisplatin and paclitaxel.The c/EBPß binding site in the COL11A1 promoter was identified as the major determinant of cisplatin- and paclitaxel-induced COL11A1 expression. Immunoprecipitation and immunofluorescence showed that in resistant cells, Akt and PDK1 were highly expressed and that anticancer drugs enhanced binding activity between COL11A1 and PDK1 binding and attenuated PDK1 ubiquitination and degradation. Conversely, chemosensitive cells showed decreased activity of COL11A1 binding to PDK1 and increased PDK1 ubiquitination, which were reversed by COL11A1 overexpression. Analysis of 104 EOC patients showed that high COL11A1 mRNA levels are significantly associated with poor chemoresponse and clinical outcome.
Assuntos
Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Colágeno Tipo XI/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Cisplatino/farmacologia , Colágeno Tipo XI/biossíntese , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/biossíntese , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ubiquitinação/efeitos dos fármacosRESUMO
Tumor necrosis factor (TNF)-α is a pleiotropic cytokine that triggers cell proliferation, cell death, or inflammation. Besides its cytotoxic effect on cancer cells, TNF-α exerts tumor promoting activity. Aberrant TNF-α signaling promotes cancer cell motility, invasiveness, and enhances cancer metastasis. Exaggerated tumor cell migration, invasion, and metastasis by TNF-α has been attributed to the activation of NF-κB signaling. It is yet to be elucidated if other signaling pathways and effector molecules are involved in TNF-α-induced cancer cell migration and metastasis. Expression of C/EBPß, a transcription factor involved in metabolism, inflammation, and cancer, is increased upon TNF-α treatment. TNF-α induces C/EBPß expression by enhancing its transcription and protein stability. Activation of p38 MAPK, but not NF-κB or JNK, is responsible for TNF-α-induced stabilization of C/EBPß protein. C/EBPß is involved in TNF-α-induced cancer cell migration. Knockdown of C/EBPß inhibits TNF-α-induced cell migration, while overexpression of C/EBPß increases migration of cancer cells. C/EBPß is translated into transcriptional activator LAP1 and LAP2 and transcriptional repressor LIP utilizing alternative in-frame translation start sites. Despite TNF-α induces expression of all three isoforms, LAP1/2, but not LIP, promote cancer cell migration. TNF-α induced MMP1/3 expression, which was abrogated by C/EBPß knockdown or p38 MAPK inhibition. MMP inhibitor or knockdown of MMP1/3 diminished TNF-α- and C/EBPß-induced cell migration. Thus, C/EBPß mediates TNF-α-induced cancer cell migration by inducing MMP1/3 expression, and may participate in the regulation of inflammation-associated cancer metastasis.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Metaloproteinase 1 da Matriz/genética , Neoplasias/genética , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 1 da Matriz/biossíntese , NF-kappa B/genética , Metástase Neoplásica , Neoplasias/patologia , Regiões Promotoras Genéticas , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Quercetin is one of the most abundant polyphenolic flavonoids found in fruits and vegetables and has anti-oxidative and anti-obesity effects. Because the small intestine is a major absorptive organ of dietary nutrients, it is likely that highly concentrated food constituents, including polyphenols, are present in the small intestinal epithelial cells, suggesting that food factors may have a profound effect in this tissue. To identify novel targets of quercetin in the intestinal enterocytes, mRNA profiling using human intestinal epithelial Caco-2 cells was performed. We found that mRNA levels of some apolipoproteins, particularly apolipoprotein B (apoB), are downregulated in the presence of quercetin. On the exposure of Caco-2 cells to quercetin, both mRNA and protein levels of apoB were decreased. Promoter analysis of the human apoB revealed that quercetin response element is localized at the 5'-proximal promoter region, which contains a conserved CCAAT enhancer-binding protein (C/EBP)-response element. We found that quercetin reduces the promoter activity of apoB, driven by the enforced expression of C/EBPß. Quercetin had no effect on either mRNA or protein levels of C/EBPß. In contrast, we found that quercetin inhibits the transcriptional activity of C/EBPß but not its recruitment to the apoB promoter. On the exposure of Caco-2 cells to quercetin 3-O-glucuronide, which is in a cell-impermeable form, no notable change in apoB mRNA was observed, suggesting an intracellular action of quercetin. In vitro interaction experiments using quercetin-conjugated beads revealed that quercetin binds to C/EBPß. Our results describe a novel regulatory mechanism of transcription of apolipoprotein genes by quercetin in the intestinal enterocytes.
Assuntos
Apolipoproteínas B/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Quercetina/farmacologia , Apolipoproteínas B/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Células CACO-2 , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ativação Transcricional/efeitos dos fármacosRESUMO
MicroRNA-191 (miR-191), a small non-coding RNA, is involved in disease development and cancer diagnosis and prognosis. However, how miR-191 functions in colorectal cancer remains largely unclear. In this study, we show that miR-191 is highly expressed in colon tumor tissues, and that inhibition of miR-191 leads to decreased cell growth, proliferation and tumorigenicity in a xenograft model. Overexpression of miR-191 in colorectal cancer cell lines alters cell cycle progression and cell resistance to 5-Fu induced cell apoptosis. Mechanistic studies demonstrated that miR-191 directly binds to the 3'UTR of the C/EBPß mRNA and mediates a decrease in the mRNA and protein expression of C/EBPß. We further showed that C/EBPß induces growth arrest in a colorectal cancer cell line and that its expression is negatively correlated with the miR-191 level in patient samples. Our findings suggest that miR-191 may be a potential gene therapy target for the treatment of colorectal cancer.