Early Growth Response Gene Research Articles

Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation. However, their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The present study was conducted to understand the role of EGR mediated regulation of VEGF A and FGF 2 signaling in buffalo luteal cells. Towards this goal, luteal cells were cultured and treated with VEGF A and FGF 2 and the mRNA expression pattern of EGR family members were documented. The EGR 1 message was found to be up-regulated in luteal cells of buffalo at 72 hours of culture. The functional validation of EGR 1 gene was accomplished by knocking out (KO) of EGR 1 in cultured luteal cells by CRISPR/Cas9 mediated gene editing technology. The EGR 1 KO cells were then cultured and stimulated with VEGF A and FGF 2. It was observed that VEGF A and FGF 2 induced angiogenesis, cell proliferation and steroidogenesis in wild type luteal cells, whereas the response of the growth factors was attenuated in the EGR 1 KO cells. Taken together our study provides evidence convincingly that both VEGF and FGF mediate their biological action through a common intermediate, EGR 1, to regulate corpus luteum function of buffalo.

A Praziquantel Treatment Study of Immune and Transcriptome Profiles in Schistosoma haematobium-Infected Gabonese Schoolchildren.

BackgroundAlthough Schistosoma haematobium infection has been reported to be associated with alterations in immune function, in particular immune hyporesponsiveness, there have been only few studies that have used the approach of removing infection by drug treatment to establish this and to understand the underlying molecular mechanisms.MethodsSchistosoma haematobium-infected schoolchildren were studied before and after praziquantel treatment and compared with uninfected controls. Cellular responses were characterized by cytokine production and flow cytometry, and in a subset of children RNA sequencing (RNA-Seq) transcriptome profiling was performed.ResultsRemoval of S haematobium infection resulted in increased schistosome-specific cytokine responses that were negatively associated with CD4+CD25+FOXP3+ T-cells and accompanied by increased frequency of effector memory T-cells. Innate responses to Toll like receptor (TLR) ligation decreased with treatment and showed positive association with CD4+CD25+FOXP3+ T-cells. At the transcriptome level, schistosome infection was associated with enrichment in cell adhesion, whereas parasite removal was associated with a more quiescent profile. Further analysis indicated that alteration in cellular energy metabolism was associated with S haematobium infection and that the early growth response genes 2 and 3 (EGR 2 and EGR3), transcription factors that negatively regulate T-cell activation, may play a role in adaptive immune hyporesponsiveness.ConclusionsUsing a longitudinal study design, we found contrasting effects of schistosome infection on innate and adaptive immune responses. Whereas the innate immune system appears more activated, the adaptive immunity is in a hyporesponsive state reflected in alterations in CD4+CD25+FOXP3+ T-cells, cellular metabolism, and transcription factors involved in anergy.

Early Growth Response Genes Increases Rapidly After Mechanical Overloading and Unloading in Tendon Constructs.

Tendon cells exist in a dense extracellular matrix and mechanical loading is important for the strength development of this matrix. We therefore use a three-dimensional (3D) culture system for tendon formation in vitro. The objectives of this study were to elucidate the temporal expression of tendon-related genes during the formation of artificial tendons in vitro and to investigate if early growth response-1 (EGR1), EGR2, FOS, and cyclooxygenase-1 and -2 (PTGS1 and PTGS2) are sensitive to mechanical loading. First, we studied messenger RNA (mRNA) levels of several tendon-related genes during formation of tendon constructs. Second, we studied the mRNA levels of, for example, EGR1 and EGR2 after different degrees of loading; dynamic physiologic-range loading (2.5% strain), dynamic overloading (approximately 10% strain), or tension release. The gene expression for tendon-related genes (i.e., EGR2, MKX, TNMD, COL3A1) increased with time after seeding into this 3D model. EGR1, EGR2, FOS, PTGS1, and PTGS2 did not respond to physiologic-range loading. But overloading (and tension release) lead to elevated levels of EGR1 and EGR2 (p ≤ 0.006). FOS and PTGS2 were increased after overloading (both p < 0.007) but not after tension release (p = 0.06 and 0.08). In conclusion, the expression of tendon-related genes increases during the formation of artificial tendons in vitro, including EGR2. Furthermore, the gene expression of EGR1 and EGR2 in human tendon cells appear to be sensitive to overloading and unloading but did not respond to the single episode of physiologic-range loading. These findings could be helpful for the understanding of tendon tensional homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:173-181, 2020.

HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation.

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.

EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

BackgroundRoles of cancer stem cells and early growth response gene 1 (Egr1) in carcinogenesis have been extensively studied in lung cancer. However, the role of Egr1 in the metastasis of lung cancer remains undetermined, especially in regard to stem cell-related pathways.MethodsEgr1, osteopontin (OPN) and Oct4 expression in human lung cancer was determined by performing immunohistochemistry. Immunoblotting, ELISA, luciferase reporter assay, chromatin immunoprecipitation assay and RT-PCR were performed to validate the regulation of Oct4-Egr1-OPN axis. Moreover, the effect of Oct4-Egr1-OPN axis on lung cancer progression was evaluated by cell migration assay and mice study.ResultsWe detected Oct4, Egr1, and OPN expression in clinical specimens from 79 lung cancer patients, including 72 adenocarcinomas and 7 squamous cell carcinomas. High expression of Oct4, Egr1, and OPN accounted for 53, 51, and 57% of the patients, respectively. All of the three biomarkers were positively correlated in clinical human lung cancer. Patients with high expression of OPN were significantly associated with shorter disease-free survivals than those with low expression of OPN (p < 0.05). In lung cancer cells, Oct4 transactivated the Egr1 promoter and upregulated Egr1 expression. In a human lung cancer xenograft model, Oct4-overexpressing tumors expressed elevated levels of Egr1. Furthermore, overexpression of Oct4 in lung cancer cells increased the metastatic potential.ConclusionsEgr1 exerts a promoting effect on cancer metastasis in Oct4-overexpressing lung cancer. Thus, therapeutic strategies targeting the Oct4/Egr1/OPN axis may be further explored for the treatment of lung cancer, especially when lung cancer is refractory to conventional treatment due to cancer stem cells.

Sensory cortex wiring requires preselection of short- and long-range projection neurons through an Egr-Foxg1-COUP-TFI network

The bimodal requisite for a genetic program and external stimuli is a key feature of sensory circuit formation. However, the contribution of cell-intrinsic codes to directing sensory-specific circuits remains unknown. Here, we identify the earliest molecular program that preselects projection neuron types in the sensory neocortex. Mechanistically, Foxg1 binds to an H3K4me1-enriched enhancer site to repress COUP-TFI, where ectopic acquisition of Foxg1 in layer 4 cells transforms local projection neurons to callosal projection neurons with pyramidal morphologies. Removal of Foxg1 in long-range projection neurons, in turn, derepresses COUP-TFI and activates a layer 4 neuron-specific program. The earliest segregation of projection subtypes is achieved through repression of Foxg1 in layer 4 precursors by early growth response genes, the major targets of the transforming growth factor-β signaling pathway. These findings describe the earliest cortex-intrinsic program that restricts neuronal connectivity in sensory circuits, a fundamental step towards the acquisition of mammalian perceptual behavior.

Egr-1 transcriptionally activates protein phosphatase PTP1B to facilitate hyperinsulinemia-induced insulin resistance in the liver in type 2 diabetes.

During the development of type 2 diabetes mellitus (T2DM), hyperinsulinemia is the earliest symptom. It is believed that long-term high insulin stimulation might facilitate insulin resistance in the liver, but the underlying mechanism remains unknown. Herein, we report that hyperinsulinemia could induce persistent early growth response gene-1 (Egr-1) activation in hepatocytes, which provides negative feedback inhibition of insulin sensitivity by inducing the expression of protein tyrosine phosphatase-1B (PTP1B). Deletion of Egr-1 in the liver remarkably decreases glucose production, thus improving systemic glucose tolerance and insulin sensitivity. Mechanistic analysis indicates that Egr-1 inhibits insulin receptor phosphorylation by directly activating PTP1B transcription in the liver. Our results reveal the molecular mechanism by which hyperinsulinemia accelerates insulin resistance in hepatocytes during the progression of T2DM.

Overexpression of Ras-Related C3 Botulinum Toxin Substrate 2 Radiosensitizes Melanoma Cells In Vitro and In Vivo.

Radioresistance is the major obstacle in the radiotherapy of the malignant melanoma. Thus, it is of importance to increase the radiosensitivity of melanoma cells. In the present study, the radioresistant melanoma cell line OCM-1 with inducible overexpression of Ras-related C3 botulinum toxin substrate 2 was established based on a radiation-inducible early growth response gene (Egr-1) promoter. The effects of Ras-related C3 botulinum toxin substrate 2 overexpression on the radiosensitivity of melanoma cells exposed to either X-rays or carbon ion beams were evaluated in cultured cells as well as xenograft tumor models. In addition, both reactive oxygen species yield and the NADPH oxidase activity were measured in the irradiated melanoma cells. It was found that the radiation-inducible overexpression of Ras-related C3 botulinum toxin substrate 2 sensitized the melanoma cells to both X-rays and carbon ion irradiation by enhancing the NADPH oxidase activity and the subsequent reactive oxygen species production. Besides, the overexpression of Ras-related C3 botulinum toxin substrate 2 enhanced the tumor-killing effect of radiotherapy in xenograft tumors significantly. The results of this study indicate that Ras-related C3 botulinum toxin substrate 2 is promising in increasing the radiosensitivity of melanoma cells, which provides experimental evidence and theoretical basis for clinical radiosensitization of the malignant melanoma.

Cellular microRNA bta-miR-2361 inhibits bovine herpesvirus 1 replication by directly targeting EGR1 gene

Bovine herpesvirus 1 (BHV-1) is an economically important pathogen of cattle and has led to significant consequences on the cattle industry worldwide. MicroRNAs (miRNAs) are a class of regulators that play critical roles in virus and host interaction. However, the roles of host miRNAs in BHV-1 infection remain largely unclear. In this study, a set of differentially expressed miRNAs by small RNA deep sequencing were analyzed in the Madin-Darby Bovine Kidney Cells (MDBK) infected with BHV-1 after 12 h, 24 h and 48 h post-infection compared to mock infection, and it was confirmed that bta-miR-2361 was significantly down-regulated. Moreover, bta-miR-2361 mimics transfection could inhibit BHV-1 replication. Combined with up-regulated genes from BHV-1-infected MDBK cells by deep RNA-sequencing and predicted by bioinformatics tools, early growth response 1 (EGR1) was putative target of bta-miR-2361. Furthermore, EGR1 was up-regulated during BHV-1 infection, and overexpression of EGR1 promoted BHV-1 replication whereas knockdown of EGR1 had the opposite effects. Subsequently, the target association between bta-miR-2361 and 3′UTR of EGR1 was further validated using a dual-luciferase reporter assay. In addition, overexpression of bta-miR-2361 resulted in decreased EGR1 mRNA and protein levels. Further mechanistic study showed that EGR1 stimulated BHV-1 UL46 promoter activity, but overexpression of bta-miR-2361 suppressed the production of UL46 gene. Collectively, this is the first study to reveal that bta-miR-2361 as a novel host factor regulates BHV-1 replication via directly targeting the EGR1 gene, which is a transcription factor that regulates viral UL46 gene of BHV-1. These results provide further insight into the study of BHV-1 pathogenesis.

MicroRNA-191-5p promotes the development of osteosarcoma via targeting EGR1 and activating the PI3K/AKT signaling pathway.

MicroRNA-191 (miR-191) has been reported to be abnormally expressed in human cancers and other diseases. The function of miR-191 was contradictory in different cancers. In the present study, we confirmed the specific function of miR-191-5p in osteosarcoma (OS). The effects of miR-191-5p on cellular behaviors of OS cells were investigated through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assay. The quantitative Real-time-polymerase chain reaction (qRT-PCR) was applied to examine the expressions of miR-191-5p and early growth response gene 1 (EGR1). Western blot and immunocytochemical assay were used to detect the protein expression of EGR1. The binding relationship between miR-191-5p and EGR1 was confirmed by Dual-Luciferase reporter gene assay. Xenograft tumor formation assay was conducted to examine the in vivo effect of miR-191-5p on tumor growth of OS. MiR-191-5p was upregulated in OS tissues, which was related to poor prognosis of OS patients. Moreover, miR-191-5p promoted cell proliferation, migration and invasion by regulating EGR1 in OS. Furthermore, EGR1 was downregulated in OS tissues, which was associated with poor prognosis of OS patients. MiR-191-5p was found to promote epithelial-mesenchymal transition (EMT) and PI3K/AKT pathway, thus promoting the development of OS. MiR-191-5p promoted the development of OS via targeting EGR1 and positively regulated the PI3K/AKT signaling pathway.

Transcriptional Regulation of Early Growth Response Gene-1 (EGR1) is Associated with Progression of Nonalcoholic Fatty Liver Disease (NAFLD) in Patients with Insulin Resistance.

BackgroundThe occurrence of nonalcoholic fatty liver disease (NAFLD) is closely related to type 2 diabetes, especially in patients with insulin resistance. The purpose of this research was to elucidate the major genes and transcriptional regulation of insulin resistance in the progression of NAFLD.Material/MethodsWe downloaded the gene expression matrix of GSE89632 from Gene Expression Omnibus. Then the principal component analysis was used to identify whether the samples were clustered. Differentially expressed genes were identified by limma R package. Enrichment analysis and protein-protein interaction network was used to find potential function and screening hub genes. We further used ChIP-seq data from ENCODE to predict the transcriptional regulation of hub genes. Finally, we verified the functions of hub genes with clinical information.ResultsThese hub genes were significantly enriched in “response to insulin”, “response to glucose”, and “fat cell differentiation”. ChIP-seq data showed that EGR1 (early growth response gene-1) may play an important role in the transcriptional regulation of SOCS1 (suppressor of cytokine signaling 1), SOCS3 (suppressor of cytokine signaling 3), and Fos gene family in the liver, as the low expression of EGR1 in patients with insulin resistance may promote the occurrence and development of NAFLD. Similarly, correlation analysis showed that EGR1 was positively correlated with the expression of SOCS1, SOCS3, and the genes of Fos gene family, and EGR1 was negatively correlated with the degree of steatosis.ConclusionsNewly identified hub genes and their transcriptional regulation may promote understanding of the molecular mechanisms underlying insulin resistance related to the progression of NAFLD and provide a new therapy target and biomarkers.

The anti-androgenic effects of cypermethrin mediated by non-classical testosterone pathway activation of mitogen-activated protein kinase cascade in mouse Sertoli cells

Previous studies have demonstrated that the anti-androgenic effects of cypermethrin (CYP) are associated with testosterone (T) - related signaling pathway. This study was to investigate the effects of CYP on mouse Sertoli cells (TM4) and clarify whether the mechanisms were mediated by non-classical T signaling pathway activating mitogen-activated protein kinase (MAPK) cascade. The Cell Counting Kit 8 (CCK8) and Real-Time Cell Analysis iCELLigence (RTCA-iCELLigence) system were performed to detect the effects of 10 μM, 20 μM, 40 μM and 80 μM CYP on the viability and proliferation of TM4. The mammalian two hybrid assay, quantitative Real-Time PCR (qRT-PCR) and western blot were conducted to analyze the key genes and proteins involved in T-mediated MAPK signaling pathway. CYP was found to inhibit the viability and proliferation of TM4. Additionally, CYP disturbed the functions of Sertoli cells by inhibiting inhibin B (INH B) expression and facilitating androgen binding protein (ABP) and transferrin (TF) expression. Moreover, CYP suppressed the interaction of AR and Src kinase and inhibited androgen-mediated phosphorylation of Src, epidermal growth factor receptor (EGFR), extracellular-regulated kinase1/2 (ERK1/2) and transcription factor cAMP response element binding protein (CREB). Furthermore, the androgen-induced mRNA and protein expression of CREB-regulated gene early growth response factor (Egr1) decreased after treated with CYP. It is indicated that CYP inhibits the viability and proliferation of Sertoli cells and non-classical T signaling pathway activation of MAPK cascade is involved in anti-androgenic effect of CYP. This study provides a novel insight into the CYP-induced reproductive toxicity.

Acoel-regeneration regulatory landscapes

Regeneration Some animals, including some types of worms, can undergo whole-body regeneration and replace virtually any missing cell type. Gehrke et al. sequenced and assembled the genome of Hofstenia miamia , a regenerative acoel worm species (see the Perspective by Alonge and Schatz). They identified a variable motif corresponding to regulation of the early growth response ( egr ) gene that was involved in regeneration. RNA interference experiments and validation in a second species showed that the protein Egr is a pioneer factor that stimulates regeneration. Science , this issue p. [eaau6173][1]; see also p. [1152][2] [1]: /lookup/doi/10.1126/science.aau6173 [2]: /lookup/doi/10.1126/science.aaw6258

CD4+CD25−LAG3+ regulatory T cells in humoral immunity

AbstractRegulatory T cells (Tregs) are necessary for the maintenance of peripheral immune tolerance. These Tregs are divided into two main subsets: thymus‐derived Tregs and peripherally‐derived Tregs. As a thymus‐derived Treg subset, CD4+CD25+ Tregs (CD25+ Tregs) that express the transcription factor, Forkhead box P3 (Foxp3), have been extensively studied. Type 1 regulatory T cells are representative Foxp3‐independent peripherally‐derived Tregs that produce a large amount of the inhibitory cytokine, interleukin (IL)‐10, and suppress immune responses through IL‐10 production. Accumulating evidence has shown that lymphocyte activation gene 3 (LAG3) is a reliable cell surface marker for type 1 regulatory T cells. Peripherally‐derived CD4+CD25−LAG3+ Tregs (LAG3+ Tregs) produce a large amount of IL‐10 and suppress colitis in a mouse model in an IL‐10‐dependent manner. LAG3+ Tregs characteristically express transcriptional factor early growth response gene 2 (Egr2), and ectopic expression of Egr2 conferred a suppressive function to CD4+ T cells. Intriguingly, polymorphism of the EGR2 gene is associated with susceptibility to systemic lupus erythematosus characterized by a wide spectrum of auto‐antibodies, and mice lacking Egr2 specifically in lymphocytes develop a lupus‐like autoimmune disease. We recently reported that Egr2‐expressing LAG3+ Tregs effectively suppress humoral immune responses in a transforming growth factor β3‐dependent manner. In addition, transforming growth factor β3 and IL‐10 synergistically regulate Toll‐like receptor‐mediated humoral immune responses. In the present review, we focus on the role of LAG3+ Tregs in humoral immunity, and also discuss its therapeutic potential for the treatment of autoimmune diseases.

Involvement of CTCF in transcription regulation of EGR1 at early G1 phase as an architecture factor

Early growth response 1 (EGR1) is a transcription factor and regulates cellular processes such as proliferation, differentiation, and apoptosis. The expression of EGR1 is rapidly induced in response to several stimuli, and it activates the expression of downstream target genes involved in signaling cascades. EGR1 gene is also known to be transcribed in early G1 phase. However, the regulation of EGR1 transcription in early G1 phase is not clarified well. Here we found that CCCTC-binding factor (CTCF), a chromatin binding protein, is required to transcribe EGR1 gene at the onset of early G1 phase. We found that CTCF mediated the formation of higher-order chromatin structures among CTCF binding sites located in the EGR1 locus. Disruption of the CTCF-dependent higher-order chromatin structure using nuclease-dead Cas9 (dCas9)-mediated interference reduced the EGR1 transcription in early G1 phase. Collectively, we propose that CTCF has functional roles for the temporal expression of EGR1 in early G1 phase through regulation of higher-order chromatin structure organization.

Corrigendum: Early Growth Response Gene-2 Is Essential for M1 and M2 Macrophage Activation and Plasticity by Modulation of the Transcription Factor CEBPβ.

[This corrects the article DOI: 10.3389/fimmu.2018.02515.].

The Netrin-1 - Neogenin Axis Regulates Hematopoietic Stem Cell Dormancy and Function with Implications for Stem Cell Ageing

Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors that differentiate into lineage-committed progenitors and subsequently mature cells. Recently, we explored the molecular signatures employed by hematopoietic-stem-cells (HSCs) during differentiation by performing quantitative proteome, transcriptome (RNA-seq) and whole genome DNA methylation analyses on dormant and active HSCs as well as multipotent progenitors populations (MPP) 1-4 (Cabezas-Wallscheid et al., Cell 2017; Cabezas-Wallscheid et al., Cell Stem Cell 2014; Klimmeck et al., Stem Cell Reports 2014; Lipka et al., Cell Cycle 2014).By exploiting these datasets, we found the DCC-like cell surface receptor Neogenin (Neo) almost exclusively expressed in CD34negCD150+CD48negLSK HSCs with an even higher expression in dormant HSCs. In addition, Neo expression has been reported to be robustly upregulated in aged HSCs (Sun et al., Cell Stem Cell 2015). The Neogenin receptor can bind different neuronal guidance molecules and can function as a BMP co-receptor. Based on this, we hypothesized that Neo and its ligands may preserve HSC dormancy and function. To investigate its role, we analyzed HSCs isolated from Neogenin-mutant mice during homeostasis and in reconstitution assays after transplantation. Neogenin-mutant HSCs show initially a competitive repopulation advantage in chimeras compared to control cells, which was associated with reduced stem cell quiescence. In agreement, RNA-seq analysis of Neogenin-mutant HSCs revealed reduced expression of dormancy related factors including the Egr1 transcription factor encoded by the early growth response gene. In contrast to the initial phases, Neogenin-mutant mice presented reduced HSC numbers associated with massively reduced reconstitution potential in 15 months old aged chimeras, as well as a myeloid differentiation bias. Collectively, these data suggest a role for Neo in preserving HSC dormancy and preventing their premature ageing.With regard to ligands of the Neo receptor, only stimulation of cultured HSCs with the axon guidance molecule Netrin-1, but not with other known Neogenin ligands affected gene expression and cellular function of HSCs. This effect of Netrin-1 was absent in Neogenin-mutant HSC demonstrating specificity. In support of the hypothesis that Netrin-1- Neo signaling may preserve HSC function, treatment of cultured HSCs with Netrin-1 alone increased the reconstitution capabilities of HSCs after transplantation compared to untreated control cells. Taken together, our results identify the Neogenin receptor as a novel player important for promoting HSC maintenance through dormancy, while its inhibition leads to exhaustion and loss of HSC self-renewal capacity upon ageing. The here identified Netrin-1- Neo axis also raise the possibility that nerves growing into the bone marrow may control HSC dormancy and function by the production of the axon guidance molecule Netrin-1. DisclosuresNo relevant conflicts of interest to declare.

Early Growth Response Gene-2 Is Essential for M1 and M2 Macrophage Activation and Plasticity by Modulation of the Transcription Factor CEBPβ.

The process of macrophage polarization is involved in many pathologies such as anti-cancer immunity and autoimmune diseases. Polarized macrophages exhibit various levels of plasticity when M2/M(IL-4) macrophages are reprogrammed into an M1-like phenotype following treatment with IFNγ and/or LPS. At the same time, M1 macrophages are resistant to reprogramming in the presence of M2-like stimuli. The molecular mechanisms responsible for the macrophages polarization, plasticity of M2 macrophages, and lack of plasticity in M1 macrophages remain unknown. Here, we explored the role of Egr2 in the induction and maintenance of macrophage M1 and M2 polarization in the mouse in vitro and in vivo models of inflammation. Egr2 knockdown with siRNA treatment fail to upregulate either M1 or M2 markers upon stimulation, and the overexpression of Egr2 potentiated M1 or M2 marker expression following polarization. Polarisation with M2-like stimuli (IL-4 or IL-13) results in increased Egr2 expression, but macrophages stimulated with M1-like stimuli (IFNγ, LPS, IL-6, or TNF) exhibit a decrease in Egr2 expression. Egr2 was critical for the expression of transcription factors CEBPβ and PPARγ in M2 macrophages, and CEBPβ was highly expressed in M1-polarized macrophages. In siRNA knockdown studies the transcription factor CEBPβ was found to negatively regulate Egr2 expression and is likely to be responsible for the maintenance of the M1-like phenotype and lack plasticity. During thioglycolate-induced peritonitis, adoptively transferred macrophages with Egr2 knockdown failed to become activated as determined by upregulation of MHC class II and CD86. Thus, our study indicates that Egr2 expression is associated with the ability of unstimulated or M2 macrophages to respond to stimulation with inflammatory stimuli, while low levels of Egr2 expression is associated with non-responsiveness of macrophages to their activation.

Induction of early growth response gene 1 (EGR1) by endoplasmic reticulum stress is mediated by the extracellular regulated kinase (ERK) arm of the MAPK pathways

Endoplasmic reticulum (ER) stress activates three principal signaling pathways, collectively known as the unfolded protein response, leading to translational and transcriptional control mechanisms that dictate the cell's response as adaptive or apoptotic. The present study illustrates that for HepG2 human hepatocellular carcinoma cells the signaling pathways triggered by ER stress extend beyond the three principal pathways to include mitogen-activated protein kinase (MAPK) signaling, leading to activation of transcription from the early growth response 1 (EGR1) gene. Analysis provided evidence for a SRC-RAS-RAF-MEK-ERK cascade mechanism that leads to enhanced phosphorylation of the transcription factor ELK1. ELK1 and serum response factor (SRF) are constitutively bound to the EGR1 promoter and are phosphorylated by nuclear localized ERK. The promoter abundance of both phospho-SRF and phopsho-ELK1 was increased by ER stress, but the SRF phosphorylation was transient. Knockdown of ELK1 had little effect on the basal EGR1 mRNA content, but completely blocked the increase in response to ER stress. Conversely, knockdown of SRF suppressed basal EGR1 mRNA content, but had only a small effect on the induction by ER stress. This research highlights the importance of MAPK signaling in response to ER stress and identifies ELK1 as a transcriptional mediator and the EGR1 gene as a target.