RESUMO
Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-ß and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-ß signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Macrófagos/imunologia , Receptor Notch1/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Ativação de Macrófagos , Macrófagos/citologia , Camundongos , Camundongos Knockout , Receptor Notch1/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genética , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Células U937RESUMO
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
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.