RESUMO
Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.
Assuntos
Interleucina-4 , Lipopolissacarídeos , Camundongos , Animais , Interleucina-4/metabolismo , Lipopolissacarídeos/metabolismo , Ligantes , Epigenômica , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Epigênese Genética , NF-kappa B/metabolismoRESUMO
Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.
Assuntos
Células Matadoras Naturais , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Animais , Camundongos , Humanos , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Anergia Clonal/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/patologia , Camundongos Endogâmicos C57BLRESUMO
Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.
Assuntos
Polaridade Celular/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Epigênese Genética/genética , Macrófagos/citologia , Fator de Transcrição STAT6/metabolismo , Ativação Transcricional/genética , Animais , Mapeamento Cromossômico , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Humanos , Interleucina-4/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Domínios e Motivos de Interação entre Proteínas/genética , Fator de Transcrição STAT6/genética , Transcriptoma/genéticaRESUMO
Alternative polarization of macrophages induced by IL-4 is important for homeostasis and tissue repair. Downstream from IL-4 receptor signaling, STAT6 activation is transient, but induces stable transcriptional changes. These data suggest that STAT6 induces second messengers to carry out the alternative transcriptional program. In this issue of Genes & Development, Daniel and colleagues (pp. 1474-1492) identify EGR2 as a downstream regulator of STAT6 with broad functionality that further induces many transcription factors associated with alternative polarization. Identification of high EGR2 expression in a subset of mouse and human alveolar macrophages further highlights EGR2 as a conserved marker of alternatively activated macrophages.
Assuntos
Ativação de Macrófagos , Macrófagos , Animais , Proteína 2 de Resposta de Crescimento Precoce , Camundongos , Fator de Transcrição STAT6 , Transdução de SinaisRESUMO
Our laboratory has a long-standing research interest in understanding how lipid-activated transcription factors, nuclear hormone receptors, contribute to dendritic cell and macrophage gene expression regulation, subtype specification, and responses to a changing extra and intracellular milieu. This journey in the last more than two decades took us from identifying target genes for various RXR heterodimers to systematically mapping nuclear receptor-mediated pathways in dendritic cells to identifying hierarchies of transcription factors in alternative polarization in macrophages to broaden the role of nuclear receptors beyond strictly ligand-regulated gene expression. We detail here the milestones of the road traveled and draw conclusions regarding the unexpectedly broad role of nuclear hormone receptors as epigenomic components of dendritic cell and macrophage gene regulation as we are getting ready for the next challenges.
Assuntos
Epigenômica , Receptores Citoplasmáticos e Nucleares , Humanos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Regulação da Expressão Gênica , Macrófagos/metabolismo , Fatores de TranscriçãoRESUMO
Pelvic organ prolapse (POP) is a common disorder among women that negatively affects women's quality of life. Early growth response 2 (EGR2) is a transcription factor that regulates cell growth. The present study aimed to explore the role of EGR2 in POP progression and provided a new target for the treatment and prevention of POP. Firstly, we extracted primary vaginal anterior wall fibroblasts from POP tissues and non-POP tissues and then constructed an EGR2-silencing lentivirus for further study. Immunoblotting, qPCR, TUNEL assay, CCK-8 assay, dual luciferase assay, and ELISA assay were carried out. EGR2 expression was much higher in POP tissues than in control tissues, and EGR2 expression positively correlated with cytokine signaling 3 (SOCS3) expression. Knockdown of EGR2 increased cell proliferation, upregulated PCNA expression, and reduced apoptosis in POP fibroblasts. Moreover, we found that the knockdown of EGR2 increased COL1A1, COL3A1, and Elastin expression and decreased MMP2 and MMP9 activities, and knockdown of EGR2 increased TGF-ß/Smad pathway activity in POP fibroblasts. Interestingly, the results of dual luciferase assay demonstrated that EGR2 was able to increase SOCS3 transcriptional activity. EGR2 knockdown alleviated the apoptosis of POP fibroblasts by reducing SOCS3 expression and improving the proliferation and collagen synthesis of POP fibroblasts. Overall, our study illustrated that EGR2 was highly expressed in POP tissues, and knockdown of EGR2 alleviated apoptosis and reduced matrix degradation in POP fibroblasts. This study might provide a new insight into the pathogenesis of POP.
Assuntos
Prolapso de Órgão Pélvico , Qualidade de Vida , Feminino , Humanos , Transdução de Sinais , Prolapso de Órgão Pélvico/metabolismo , Prolapso de Órgão Pélvico/patologia , Vagina/metabolismo , Vagina/patologia , Luciferases/metabolismoRESUMO
During abscisic acid (ABA) signaling, reversible phosphorylation controls the activity and accumulation of class III SNF1-RELATED PROTEIN KINASE 2s (SnRK2s). While protein phosphatases that negatively regulate SnRK2s have been identified, those that positively regulate ABA signaling through SnRK2s are less understood. In this study, Arabidopsis thaliana mutants of Clade E Growth-Regulating 1 and 2 (EGR1/2), which belong to the protein phosphatase 2C family, exhibited reduced ABA sensitivity in terms of seed germination, cotyledon greening, and ABI5 accumulation. Conversely, overexpression increased these ABA-induced responses. Transcriptomic data revealed that most ABA-regulated genes in egr1 egr2 plants were expressed at reduced levels compared with those in Col-0 after ABA treatment. Abscisic acid up-regulated EGR1/2, which interact directly with SnRK2.2 through its C-terminal domain I. Genetic analysis demonstrated that EGR1/2 function through SnRK2.2 during ABA response. Furthermore, SnRK2.2 de-phosphorylation by EGR1/2 was identified at serine 31 within the ATP-binding pocket. A phospho-mimic mutation confirmed that phosphorylation at serine 31 inhibited SnRK2.2 activity and reduced ABA responsiveness in plants. Our findings highlight the positive role of EGR1/2 in regulating ABA signaling, they reveal a new mechanism for modulating SnRK2.2 activity, and provide novel insight into how plants fine-tune their responses to ABA.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fosforilação , Ácido Abscísico/farmacologia , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Serina/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Vogt-Koyanagi-Harada (VKH) disease, a major blinding eye disease, is characterized by an autoimmune response against melanocytes in multiple organs throughout the body. Currently, the aetiology and pathogenesis of VKH disease are unclear, and the treatment strategy needs to be further optimized. The retinal pigment epithelium (RPE), a monolayer of pigmented cells of the fundus, is essential for maintaining normal visual function and is involved in both the acute and chronic stages of VKH disease. Therefore, the functions of the RPE may play a critical role in the aetiology and treatment of VKH disease. Herein, we established a human induced pluripotent stem cell (hiPSC) RPE model of VKH disease by reprogramming peripheral blood mononuclear cells (PBMCs) into iPSCs and then differentiating them into RPE cells. Patient-derived RPE cells exhibited barrier disruption, impaired phagocytosis, and depigmentation compared with those from normal controls, which was consistent with the features of VKH disease. Furthermore, a small molecular compound targeting EGR2 was found to rescue the barrier and phagocytic functions of the hiPSC-RPE cells through high-throughput virtual screening and functional studies, suggesting a promising strategy for the treatment of VKH disease.
Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome Uveomeningoencefálica , Humanos , Síndrome Uveomeningoencefálica/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Leucócitos Mononucleares , Epitélio Pigmentado da RetinaRESUMO
OST1 (open stomata 1) protein kinase plays a central role in regulating freezing tolerance in Arabidopsis; however, the mechanism underlying cold activation of OST1 remains unknown. Here, we report that a plasma membrane-localized clade-E growth-regulating 2 (EGR2) phosphatase interacts with OST1 and inhibits OST1 activity under normal conditions. EGR2 is N-myristoylated by N-myristoyltransferase NMT1 at 22°C, which is important for its interaction with OST1. Moreover, myristoylation of EGR2 is required for its function in plant freezing tolerance. Under cold stress, the interaction of EGR2 and NMT1 is attenuated, leading to the suppression of EGR2 myristoylation in plants. Plant newly synthesized unmyristoylated EGR2 has decreased binding ability to OST1 and also interferes with the EGR2-OST1 interaction under cold stress. Consequently, the EGR2-mediated inhibition of OST1 activity is released. Consistently, mutations of EGRs cause plant tolerance to freezing, whereas overexpression of EGR2 exhibits decreased freezing tolerance. This study thus unravels a molecular mechanism underlying cold activation of OST1 by membrane-localized EGR2 and suggests that a myristoyl switch on EGR2 helps plants to adapt to cold stress.
Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis , Temperatura Baixa/efeitos adversos , Proteínas Quinases/metabolismo , Proteína Fosfatase 2C/fisiologia , Aclimatação/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Resposta ao Choque Frio/genética , Resposta ao Choque Frio/fisiologia , Ativação Enzimática/genética , Ácidos Graxos Monoinsaturados/metabolismo , Congelamento , Regulação da Expressão Gênica de Plantas , Fosforilação , Plantas Geneticamente Modificadas , Processamento de Proteína Pós-Traducional/genética , Transdução de SinaisRESUMO
Although T cells can develop into an exhausted state in the tumour microenvironment, tumour infiltrating T cells (TILs) are important to control tumour growth. By analysing single cell RNA-sequencing data from human tumours, we found that the transcription factors Early Growth Response 2 (EGR2) and 3 were highly induced in TILs, but not peripheral CD8 + T cells, in multiple patient cohorts. We found that deficiency of Egr2 and 3 in T cells resulted in enhanced tumour growth and fewer TILs in mouse models. Egr2 is highly expressed together with checkpoint molecules in a proportion of CD8 + TILs and Egr2high cells exhibit better survival and proliferation than Egr2-/-Egr3-/- and Egr2low TILs. Anti-PD-1 treatment increases Egr2 expression in CD8 + TILs and reduces tumour growth, while anti-PD-1 efficacy is abrogated in the absence of Egr2 and 3. Thus, Egr2 and 3 are important for maintaining anti-tumour responses of exhausted CD8 + TILs.
Assuntos
Neoplasias , Camundongos , Animais , Humanos , Neoplasias/patologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Microambiente Tumoral , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismoRESUMO
Immunotherapy for prostate cancer (PCa) faces serious challenges. Therefore, the co-inhibitory receptors that regulate T cell function of PCa must be elucidated. Here we identified that the inhibitory receptor LAG3 was significantly induced in T cells from PCa patients. Gene array analysis revealed that insufficient ataxia telangiectasia mutated (ATM) gene expression in PCa T cells was responsible for the elevated LAG3 expression. Mechanistically, insufficient ATM expression impaired its ability to activate AMPKα signaling and CD4+ T cell functions, which further enhances the binding of the transcription factors XBP1 and EGR2 to LAG3 promoter. Reconstitution of ATM and inhibition of XBP1 or EGR2 in PCa T cells suppressed LAG3 expression and restored the effector function of CD4+ T cells from PCa. Our study revealed the mechanism of LAG3 upregulation in CD4+ T lymphocytes of PCa patients and may provide insights for the development of immunotherapeutic strategies for PCa treatment.
Assuntos
Neoplasias da Próstata , Linfócitos T , Masculino , Humanos , Linfócitos T/metabolismo , Transdução de Sinais , Regulação para Cima , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
BACKGROUND: Mutations in the Early-Growth Response 2 (EGR2) gene cause various hereditary neuropathies, including demyelinating Charcot-Marie-Tooth (CMT) disease type 1D (CMT1D), congenital hypomyelinating neuropathy type 1 (CHN1), Déjerine-Sottas syndrome (DSS), and axonal CMT (CMT2). METHODS: In this study, we identified 14 patients with heterozygous EGR2 mutations diagnosed between 2000 and 2022. RESULTS: Mean age was 44 years (15-70), 10 patients were female (71%), and mean disease duration was 28 years (1-56). Disease onset was before age 15 years in nine cases (64%), after age 35 years in four cases (28%), and one patient aged 26 years was asymptomatic (7%). All symptomatic patients had pes cavus and distal lower limbs weakness (100%). Distal lower limbs sensory symptoms were observed in 86% of cases, hand atrophy in 71%, and scoliosis in 21%. Nerve conduction studies showed a predominantly demyelinating sensorimotor neuropathy in all cases (100%), and five patients needed walking assistance after a mean disease duration of 50 years (47-56) (36%). Three patients were misdiagnosed as inflammatory neuropathy and treated with immunosuppressive drugs for years before diagnosis was corrected. Two patients presented with an additional neurologic disorder, including Steinert's myotonic dystrophy and spinocerebellar ataxia (14%). Eight EGR2 gene mutations were found, including four previously undescribed. INTERPRETATION: Our findings demonstrate EGR2 gene-related hereditary neuropathies are rare and slowly progressive demyelinating neuropathies with two major clinical presentations, including a childhood-onset variant and an adult-onset variant which may mimic inflammatory neuropathy. Our study also expands the genotypic spectrum of EGR2 gene mutations.
Assuntos
Doença de Charcot-Marie-Tooth , Neuropatia Hereditária Motora e Sensorial , Adulto , Humanos , Feminino , Criança , Masculino , Distribuição por Idade , Doença de Charcot-Marie-Tooth/genética , Mutação , Genótipo , Fenótipo , Proteína 2 de Resposta de Crescimento Precoce/genéticaRESUMO
Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs.
Assuntos
Síndrome de Rett , Camundongos , Animais , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Genes Precoces , Proteína 2 de Ligação a Metil-CpG/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Hipocampo/metabolismo , Modelos Animais de Doenças , Mamíferos/metabolismoRESUMO
Apoptosis genes Egr2, Fas and FasL are related to immune responses. However, the mechanism of these genes inducing apoptosis in fish are still not very clear. An acute hypoxia treatment (1.73 ± 0.06 mg/L) for 24 h was carried out on Japanese flounder (Paralichthys olivaceus). The increasingly dense apoptotic signals at 3 h, 6 h, 12 h by TUNEL in skeletal muscle indicated that hypoxia could quickly affect muscle growth and development. Furthermore, we concluded that the Egr2-FasL-Fas signal pathway, which was located at the upstream of apoptotic executor protein caspases, was related to the apoptosis by quantitative real-time PCR, protein concentration detection in ELISA and double gene in situ hybridization methods. The mechanism of the pathway was researched in transcription regulation and epigenetic modification by dual-luciferase reporter assay and bisulfite modified method, respectively. Egr2, as a transcription factor, could up-regulate the expression of FasL gene. And its binding site was mainly between -479 to -1 of FasL gene promoter. The 5th CpG dinucleotides (-514) methylation levels in FasL gene were significantly affected by hypoxia, and they were negatively correlated with its expressions. These suggested that the -514 site may be a very important site to regulate the FasL gene expression. Above results, we concluded that hypoxia activated the immune related signal pathway Egr2-FasL-Fas to induced skeletal muscle apoptosis to affect growth and development of Japanese flounder. The study revealed the mechanism of hypoxia induced apoptosis, which could provide a reference for fish immunity and aquaculture management.
Assuntos
Linguado , Animais , Apoptose/fisiologia , Proteína Ligante Fas/genética , Regulação da Expressão Gênica , Hipóxia/genética , Hipóxia/veterinária , Transdução de SinaisRESUMO
BACKGROUND: Myocardial infarction (MI) is characterized by coronary artery occlusion, ischemia and hypoxia of myocardial cells, leading to irreversible myocardial damage. Therefore, it is urgent to explore the potential mechanism of myocardial injury during the MI process to develop effective therapies for myocardial cell rescue. METHODS: We downloaded the GSE71906 dataset from GEO DataSets, and the R software was used to identify the differentially expressed genes (DEGs) in mouse heart tissues of MI and sham controls. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed to understand the significantly activated signaling pathways in MI. Protein-protein interaction (PPI) network was constructed to highlight the hub genes in DEGs. The Western Blot, qRT-PCR and TUNEL staining were used to explore the function of hub gene in hypoxia-induced cardiomyocytes in vitro. RESULTS: A total of 235 DEGs were identified in GSE71906 dataset. Functional enrichment analysis revealed that the upregulated genes were primarily associated with the inflammatory response and apoptosis. 20 hub genes were identified in PPI network, and the early growth response 2 (EGR2) was highlighted. In vitro. We confirmed the EGR2 was upregulated induced by hypoxia and revealed the upregulated EGR2 aggravates pro-inflammation and pro-apoptotic genes expression. In addition, EGR2 knockout mitigates hypoxia-induced inflammation and apoptosis in cardiomyocytes. CONCLUSION: The present study identified the EGR2 was a hub gene in myocardial damage during MI process, the excessive EGR2 aggravates hypoxia-induced myocardial damage by accelerating inflammation and apoptosis in vitro. Therefore, targeting EGR2 offers a potential pharmacological strategy for myocardial cell rescue in MI.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce , Infarto do Miocárdio , Miócitos Cardíacos , Animais , Apoptose , Biologia Computacional , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , Camundongos , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20-deficient embryos. Genetic lineage tracing in Krox20-/- mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve.
Assuntos
Valva Aórtica/anormalidades , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Células Endoteliais/metabolismo , Doenças das Valvas Cardíacas/embriologia , Miocárdio/metabolismo , Crista Neural/metabolismo , Animais , Valva Aórtica/citologia , Valva Aórtica/embriologia , Doença da Válvula Aórtica Bicúspide , Proteína 2 de Resposta de Crescimento Precoce/genética , Células Endoteliais/citologia , Camundongos , Camundongos Knockout , Miocárdio/citologia , Crista Neural/citologiaRESUMO
BACKGROUND: Recent studies have shown that early growth response 2 (EGR2) is highly induced in activated T cells and regulates T cell functions. In normal C57BL/6 (B6) mice, deletion of EGR2 in lymphocytes results in the development of lupus-like systemic autoimmune disease, which implies indirectly an autoimmune protective role of EGR2. Conversely, increased EGR2 gene expression is suggested to link with high risk of human lupus. In the present studies we sought to clarify the expression and inflammation regulatory role of EGR2 in murine lupus T cells directly. RESULTS: We performed RT-qPCR analysis and found a significant increase of EGR2 mRNA expression in human lupus PBMCs and in CD4+ T cells from three different murine lupus models including MRL-lpr, B6-lpr, and B6.sle123 mice at diseased stage when compared to age-matched control MRL or B6 mice. By performing intracellular flow cytometry analysis, we found that EGR2 protein expression was significantly increased in resting lupus (either MRL-lpr or B6.sle123) CD4+ T cells when compared to CD4+ T cells from their respective non-autoimmune controls. However, there was no difference of EGR2 protein expression in anti-CD3 and anti-CD28 stimulated control and lupus CD4+ T cells since there was a stronger induction of EGR2 in activated control CD4+ T cells. EGR2 expression was significantly increased in MRL-lpr mice at an age when lupus is manifested. To understand further the function of elevated EGR2 in lupus CD4+ T cells, we inhibited EGR2 with a specific siRNA in vitro in splenocytes from MRL-lpr and control MRL mice at 15 weeks-of-age. We found that EGR2 inhibition significantly reduced IFNγ production in PMA and ionomycin activated MRL-lpr lupus CD4+ T cells, but not control MRL CD4+ T cells. We also found that inhibition of EGR2 in vitro suppressed the Th1 differentiation in both MRL and MRL-lpr naïve CD4+ T cells. CONCLUSIONS: EGR2 is highly upregulated in human and murine lupus cells. Our in vitro data suggest a positive role of EGR2 in the regulation of Th1 differentiation and IFNγ production in lupus effector CD4+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Inflamação/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Proteína 2 de Resposta de Crescimento Precoce/genética , Humanos , Inflamação/imunologia , Interferon gama/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Regulação para CimaRESUMO
BACKGROUND AND PURPOSE: Mutations in the early growth response 2 gene (EGR2) cause demyelinating, but also axonal, neuropathies differing in severity and age of onset. Except for one family, all reported cases have autosomal dominant inheritance and mutations are localized within the three zinc finger (ZNF) DNA-binding domain. The aim of this study was to provide a clinical and molecular analysis of a novel recessive mutation in EGR2. METHODS: Clinical and electrophysiological assessments of three affected patients, from a consanguineous family, were performed. Genetic analyses of EGR2 were carried out by Sanger sequencing. Functional effects of clinical recessive mutations were assessed using a mammalian two-hybrid assay. RESULTS: A novel missense mutation (c.791C>T; p.P264L) in the homozygous state was detected outside the ZNF domains of the EGR2 gene. Three affected siblings presented with distal demyelinating polyneuropathy with severe sensory loss, progressive thoracolumbar scoliosis and trigeminal neuralgia. Respiratory compromise and cranial nerve dysfunction were also found. Our data indicate that the p.P264L mutation prevents interaction of EGR2 transcription factor with NAB corepressors, suggesting that a disruption of the NAB-EGR2 protein interactions can result in dramatic neuropathy. CONCLUSION: Mutations in, or next to, the R1 domain of EGR2 should be considered with extreme caution for genetic counseling, since these could cause a severe neuropathy with an autosomal recessive manner of transmission.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Animais , Homozigoto , Humanos , Mutação , Fatores de Transcrição/genéticaRESUMO
BACKGROUND/AIMS: Hirschsprung's disease (HSCR) is the most common digestive disease caused by disorders of neural crest development. Despite the known involvement of miR-140-5p in many human diseases, its biological role in Hirschsprung's disease (HSCR) remains undefined. In this study, we sought to reveal the roles of miR-140-5p in the pathogenesis of HSCR. METHODS: Quantitative real-time PCR and western blotting were used to measure the relative expression levels of miRNAs, mRNAs, and proteins in stenotic and dilated sections of the colon of 32 HSCR patients. Targets and proteins were evaluated by western blotting, and Transwell, CCK-8, and flow cytometry assays were adopted to detect the functional effects of miR-140-5p on SH-SY5Y cells. RESULTS: miR-140-5p was significantly downregulated in HSCR tissue samples with increased expression of EGR2, and knockdown of miR-140-5p inhibited cell migration and proliferation and promoted apoptosis in SH-SY5Y cell lines. EGR2 expression was inversely correlated with that of miR-140-5p in cell lines. CONCLUSIONS: miR-140-5p may influence the pathogenesis of HSCR by targeting EGR2.
Assuntos
Regulação para Baixo/fisiologia , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Doença de Hirschsprung/patologia , MicroRNAs/antagonistas & inibidores , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Regulação para Baixo/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Feminino , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
EGR2 (Early Growth Response 2) is one of the most important transcription factors involved in myelination in the peripheral nervous system. EGR2 mutations typically cause different forms of demyelinating neuropathy, that is, Charcot-Marie-Tooth type 1D (CMT1D), Dejerine-Sottas Syndrome (DSS), and Congenital Hypomyelinating Neuropathy (CHN). However, the EGR2 gene has been recently associated with an axonal phenotype (CMT2) in a large CMT family. Here, we report another CMT family exhibiting an axonal phenotype associated with a missense change (c.1235A>G, p.E412G) in the EGR2 gene. Neurological evaluation of five affected members of the family showed a classical CMT phenotype including distal muscle atrophy and weakness, absence of deep tendon reflexes, pes cavus, and scoliosis. Electrophysiological examination was consistent with a motor-sensory axonal neuropathy. Sural nerve biopsy performed in one patient showed a loss of myelinated and unmyelinated nerve fibers without de-remyelinating signs and onion bulbs. This study confirms the phenotypical heterogeneity of EGR2-related neuropathy, indicating a role for EGR2 in primary axonal degeneration.