RESUMO
The epidermal growth factor-like protein DLK2, highly homologous to DLK1, has been identified as a modulator of adipogenesis in vitro. Knocking down Dlk2 expression prevents adipogenesis of 3T3-L1 cells but enhances that of the mesenchymal cell line C3H10T1/2. The expression of Dlk2 shows two peaks along this differentiation process: the first one, in response to 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (Dex), and the second, shortly after exposure to insulin. Nothing is known about the transcriptional regulation of Dlk2 during adipogenesis. Here, we report that, during early adipogenesis of 3T3-L1 cells, Dlk2 expression is controlled independently by IBMX and Dex. We also show that KLF4, a transcription factor critical for the control of early adipogenesis, binds directly to the Dlk2 promoter and increases Dlk2 expression in response to IBMX. Overexpression of KLF4 leads to an increase in DLK2 expression levels, whereas KLF4 knockdown downregulates the transcriptional activity of the Dlk2 promoter. Finally, we demonstrate that KLF4 regulates the basal expression of Dlk2 in C3H10T1/2 cells, and it is required for the adipogenic differentiation of those cells. These results indicate that KLF4 mediates the transcriptional regulation of Dlk2 in response to IBMX during the early stages of adipogenesis.
Assuntos
Adipogenia/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fatores de Transcrição Kruppel-Like/genética , Transcrição Gênica , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3-L1 , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Inibidores de Fosfodiesterase/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: DLK2 is an EGF-like membrane protein, closely related to DLK1, which is involved in adipogenesis. Both proteins interact with the NOTCH1 receptor and are able to modulate its activation. The expression of the gene Dlk2 is coordinated with that of Dlk1 in several tissues and cell lines. Unlike Dlk1, the mouse Dlk2 gene and its locus at chromosome 17 are not fully characterized. RESULTS: The goal of this work was the characterization of Dlk2 mRNA, as well as the analysis of the mechanisms that control its basal transcription. First, we analyzed the Dlk2 transcripts expressed by several mouse cells lines and tissues, and mapped the transcription start site by 5' Rapid Amplification of cDNA Ends. In silico analysis revealed that Dlk2 possesses a TATA-less promoter containing minimal promoter elements associated with a CpG island, and sequences for Inr and DPE elements. Besides, it possesses six GC-boxes, considered as consensus sites for the transcription factor Sp1. Indeed, we report that Sp1 directly binds to the Dlk2 promoter, activates its transcription, and regulates its level of expression. CONCLUSIONS: Our results provide the first characterization of Dlk2 transcripts, map the location of the Dlk2 core promoter, and show the role of Sp1 as a key regulator of Dlk2 transcription, providing new insights into the molecular mechanisms that contribute to the expression of the Dlk2 gene.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Ilhas de CpG , Regulação da Expressão Gênica , Ordem dos Genes , Inativação Gênica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/química , RNA Interferente Pequeno , Fator de Transcrição Sp1/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica , Ativação TranscricionalRESUMO
Macrophages activated through Toll receptor triggering increase the expression of the A(2A) and A(2B) adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A(2A) and A(2B) receptors, we demonstrate that the A(2A) receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A(2B) receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1α is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A(2) receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.
Assuntos
Adenosina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/enzimologia , Fosfofrutoquinase-2/biossíntese , Receptor 4 Toll-Like/agonistas , Adenosina/genética , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Frutosedifosfatos/genética , Frutosedifosfatos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glicólise/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosfofrutoquinase-2/genética , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina , Deleção de Sequência , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.
Assuntos
Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Células 3T3 , Células 3T3-L1 , Proteínas Adaptadoras de Transdução de Sinal , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Ligação Competitiva , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Células HEK293 , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ligação Proteica , Receptor Notch1/genética , Proteínas Serrate-Jagged , Técnicas do Sistema de Duplo-HíbridoRESUMO
Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-kappaB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-alpha and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-kappaB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-kappaB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IkappaB kinase alpha/beta complex and the expression of some NF-kappaB family members. Treatment of macrophages with the gamma-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-kappaB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-alpha and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-kappaB signaling.
Assuntos
Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , NF-kappa B/imunologia , Receptor Notch1/imunologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , NF-kappa B/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
BACKGROUND: The Dlk1 gene encodes for dlk1, a transmembrane protein belonging to the EGF-like repeat-containing family. Dlk1 has been shown to act as a regulator of adipogenesis. Fc-dlk1 transgenic mice show a decrease in adipose tissue and glucose tolerance, hypertriglyceridaemia and lower insulin sensitivity. Dlk1-deficient mice show growth retardation, increased serum lipid metabolites and develop obesity. These data advocate for a role of dlk1 in the maintenance of lipid homeostasis, and suggest that dlk1 levels may influence the development of cardiovascular disease. AIM AND METHODS: In this study, we analysed whether dlk1 serum levels could be indicative of the different hormonal or metabolic status shown by two Spanish children populations (6-8 years-old), Orense and Murcia. We determined dlk1 serum levels by ELISA assay, using an antibody raised against the recombinant protein, and performed a correlation analysis against measurements of several hormonal and biochemical parameters in samples from 494 subjects. RESULTS: We found a statistically significant positive correlation between serum levels of dlk1 and those of glucose (P < 0.05), total cholesterol (P < 0.01) and high-density lipoprotein-cholesterol (HDL-C) (P < 0.01) in children from Murcia, but not from Orense's population, where dehydroepiandrosterone-sulphate (DHEA-S) levels were significantly higher (P < 0.01) and dlk1 correlated positively with insulin (P < 0.01), homeostasis model assessment (HOMA) (P < 0.01) and free fatty acids (FFA) (P < 0.05). CONCLUSIONS: dlk1 serum levels appear related to the anabolic status of the children in association with changes in the levels of DHEA-S, which have been associated with hyperinsulinaemia and diabetes. Monitoring dlk1 levels may be important to evaluate the metabolic and hormonal stage of child development.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Desenvolvimento Infantil/fisiologia , Hormônios/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/sangue , Células 3T3-L1 , Animais , Células 3T3 BALB , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio , Criança , Indicadores Básicos de Saúde , Hormônios/análise , Hormônios/metabolismo , Humanos , Camundongos , Pichia , EspanhaRESUMO
The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.