RESUMO
BACKGROUND: Atherosclerosis is an inflammatory disease in which macrophages play a crucial role. Macrophages are present in different phenotypes, with at the extremes of the spectrum the classical M1 pro-inflammatory and the alternative M2 anti-inflammatory macrophages. The neuron-derived orphan receptor 1 (NOR1), together with Nur77 and Nurr1, are members of the NR4A orphan nuclear receptor family, expressed in human atherosclerotic lesion macrophages. However, the role of NOR1 in human macrophages has not been studied yet. OBJECTIVES: To determine the expression and the functions of NOR1 in human alternative macrophages. METHODS AND RESULTS: In vitro IL-4 polarization of primary monocytes into alternative M2 macrophages enhances NOR1 expression in human but not in mouse macrophages. Moreover, NOR1 expression is most abundant in CD68+MR+ alternative macrophage-enriched areas of human atherosclerotic plaques in vivo. Silencing NOR1 in human alternative macrophages decreases the expression of several M2 markers such as the Mannose Receptor (MR), Interleukin-1 Receptor antagonist (IL-1Ra), CD200 Receptor (CD200R), coagulation factor XIII A1 polypeptide (F13A1), Interleukin 10 (IL-10) and the Peroxisome Proliferator-Activated Receptor (PPAR)γ. Bioinformatical analysis identified F13A1, IL-1Ra, IL-10 and the Matrix Metalloproteinase-9 (MMP9) as potential target genes of NOR1 in human alternative macrophages. Moreover, expression and enzymatic activity of MMP9 are induced by silencing and repressed by NOR1 overexpression in M2 macrophages. CONCLUSIONS: These data identify NOR1 as a transcription factor induced during alternative differentiation of human macrophages and demonstrate that NOR1 modifies the alternative macrophage phenotype.
Assuntos
Doenças das Artérias Carótidas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Biomarcadores/metabolismo , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/patologia , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Inativação Gênica , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-4/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Placa Aterosclerótica , Cultura Primária de Células , Interferência de RNA , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
The orphan nuclear receptor HNF4alpha and the LIM homeodomain factor Isl1 are co-expressed in pancreatic beta-cells and are required for the differentiation and function of these endocrine cells. HNF4alpha activates numerous genes and mutations in its gene are associated with maturity onset diabetes of the young. Cofactors and transcription factors that interact with HNF4alpha are crucial to modulate its transcriptional activity, since the latter is not regulated by conventional ligands. These transcriptional partners interact mainly through the HNF4alpha AF-1 module and the ligand binding domain, which contains the AF-2 module. Here, we showed that Isl1 could enhance the HNF4alpha-mediated activation of transcription of the HNF1alpha, PPARalpha and insulin I promoters. Isl1 interacted with the HNF4alpha AF-2 but also required the HNF4alpha carboxy-terminal F domain for optimal interaction and transcriptional synergy. More specifically, we found that naturally occurring HNF4alpha isoforms, differing only in their F domain, exhibited different abilities to interact and synergize with Isl1, extending the crucial transcriptional modulatory role of the HNF4alpha F domain. HNF4alpha interacted with both the homeodomain and the first LIM domain of Isl1. We found that the transcriptional synergy between HNF4alpha and Isl1 involved an increase in HNF4alpha loading on promoter. The effect was more pronounced on the rat insulin I promoter containing binding sites for both HNF4alpha and Isl1 than on the human HNF1alpha promoter lacking an Isl1 binding site. Moreover, Isl1 could mediate the recruitment of the cofactor CLIM2 resulting in a further transcriptional enhancement of the HNF1alpha promoter activity.
Assuntos
Fator 4 Nuclear de Hepatócito/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Proteínas de Ligação a DNA/metabolismo , Fator 1 Nuclear de Hepatócito/genética , Humanos , Insulina/genética , Proteínas com Domínio LIM , Proteínas com Homeodomínio LIM , Ligantes , PPAR alfa/genética , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ativação TranscricionalRESUMO
Hepatocyte Nuclear Factor 1alpha (HNF1alpha) and Hepatocyte Nuclear Factor 4alpha (HNF4alpha) are two liver-enriched transcription factors coexpressed in specific tissues where they play a crucial role through their involvement in a complex cross-regulatory network. HNF1alpha down regulates HNF4alpha-mediated activation of transcription via a direct protein-protein interaction. Here we show that HNF4alpha enhances the transcriptional activity of HNF1alpha in a DNA binding independent manner, thus indicating that it behaves as a HNF1alpha coactivator. Using mutations in the ligand binding domain (LBD) of HNF4alpha, we confirmed the involvement of the Activation Function 2 module and demonstrated the requirement of the integrity of the LBD for the interaction with HNF1alpha. Moreover, we show that HNF4alpha cooperates with p300 to achieve the highest HNF1alpha-mediated transcription rates. Our findings highlight a new way by which HNF4alpha can regulate gene expression and extend our knowledge of the complexity of the transcriptional network involving HNF4alpha and HNF1alpha.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Ligação a DNA/química , Células HeLa , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 4 Nuclear de Hepatócito , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Transativadores/metabolismo , Fatores de Transcrição/químicaRESUMO
The nuclear receptor hepatocyte nuclear factor (HNF) 4 alpha is involved in a transcriptional network and plays an important role in pancreatic beta-cells. Mutations in the HNF4 alpha gene are correlated with maturity-onset diabetes of the young 1. HNF4 alpha isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4 alpha transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic beta-cells and in the pancreatic beta-cell lines RIN m5F and HIT-T15. Expression of HNF4 alpha proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4 alpha isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1 alpha that is required for beta-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic beta-cell function.
Assuntos
Proteínas de Ligação a DNA , Ilhotas Pancreáticas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Western Blotting , Linhagem Celular , Fator 4 Nuclear de Hepatócito , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RatosRESUMO
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a nuclear receptor involved in glucose homeostasis and is required for normal beta-cell function. Mutations in the HNF4alpha gene are associated with maturity-onset diabetes of the young type 1. E276Q and R154X mutations were previously shown to impair intrinsic transcriptional activity (without exogenously supplied co-activators) of HNF4alpha. Given that transcriptional partners of HNF4alpha modulate its intrinsic transcriptional activity and play crucial roles in HNF4alpha function, we investigated the effects of these mutations on potentiation of HNF4alpha activity by p300, a key co-activator for HNF4alpha. We show here that loss of HNF4alpha function by both mutations is increased through impaired physical interaction and functional cooperation between HNF4alpha and p300. Impairment of p300-mediated potentiation of HNF4alpha transcriptional activity is of particular importance for the E276Q mutant since its intrinsic transcriptional activity is moderately affected. Together with previous results obtained with chicken ovalbumin upstream promoter-transcription factor II, our results highlight that impairment of recruitment of transcriptional partners represents an important mechanism leading to abnormal HNF4alpha function resulting from the MODY1 E276Q mutation. The impaired potentiations of HNF4alpha activity were observed on the promoter of HNF1alpha, a transcription factor involved in a transcriptional network and required for beta-cell function. Given its involvement in a regulatory signaling cascade, loss of HNF4alpha function may cause reduced beta-cell function secondary to defective HNF1alpha expression. Our results also shed light on a better structure-function relationship of HNF4alpha and on p300 sequences involved in the interaction with HNF4alpha.
Assuntos
Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 2/genética , Mutação , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Linhagem Celular , Diabetes Mellitus Tipo 2/fisiopatologia , Embrião de Mamíferos , Células HeLa , Fator 4 Nuclear de Hepatócito , Humanos , Ilhotas Pancreáticas/fisiopatologia , Rim , Fosfoproteínas/química , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
Mutations in the hepatocyte nuclear factor 4alpha (HNF-4alpha) gene are associated with one form of maturity-onset diabetes of the young (MODY1). The R154X mutation generates a protein lacking the E-domain which is required for normal HNF-4alpha functions. Since pancreatic beta-cell dysfunction is a feature of MODY1 patients, we compared the functional properties of the R154X mutant in insulin-secreting pancreatic beta-cells and non-beta-cells. The R154X mutation did not affect nuclear localisation in beta-cells and non-beta-cells. However, it did lead to a greater impairment of HNF-4a function in beta-cells compared to non-beta-cells, including a complete loss of transactivation activity and a dominant-negative behaviour. .