Early Growth Response Gene Research Articles

High expression of transcription factor EGR1 is associated with postoperative muscle atrophy in patients with knee osteoarthritis undergoing total knee arthroplasty

BackgroundMuscle atrophy is a typical affliction in patients affected by knee Osteoarthritis (KOA). This study aimed to examine the potential pathogenesis and biomarkers that coalesce to induce muscle atrophy, primarily through the utilization of bioinformatics analysis.MethodsTwo distinct public datasets of osteoarthritis and muscle atrophy (GSE82107 and GSE205431) were subjected to differential gene expression analysis and gene set enrichment analysis (GSEA) to probe for common differentially expressed genes (DEGs) and conduct transcription factor (TF) enrichment analysis from such genes. Venn diagrams were used to identify the target TF, followed by the construction of a protein-protein interaction (PPI) network of the common DEGs governed by the target TF. Hub genes were determined through the CytoHubba plug-in whilst their biological functions were assessed using GSEA analysis in the GTEx database. To validate the study, reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and Flow Cytometry techniques were employed.ResultsA total of 138 common DEGs of osteoarthritis and muscle atrophy were identified, with 16 TFs exhibiting notable expression patterns in both datasets. Venn diagram analysis identified early growth response gene-1 (EGR1) as the target TF, enriched in critical pathways such as epithelial mesenchymal transition, tumor necrosis factor-alpha signaling NF-κB, and inflammatory response. PPI analysis revealed five hub genes, including EGR1, FOS, FOSB, KLF2, and JUNB. The reliability of EGR1 was confirmed by validation testing, corroborating bioinformatics analysis trends.ConclusionsEGR1, FOS, FOSB, KLF2, and JUNB are intricately involved in muscle atrophy development. High EGR1 expression directly regulated these hub genes, significantly influencing postoperative muscle atrophy progression in KOA patients.

A Methyl-Engineered DNAzyme for Endogenous Alkyltransferase Monitoring and Self-Sufficient Gene Regulation.

The on-demand gene regulation is crucial for extensively exploring specific gene functions and developing personalized gene therapeutics, which shows great promise in precision medicines. Although some nucleic acid-based gene regulatory tools (antisense oligonucleotides and small interfering RNAs) are devised for achieving on-demand activation, the introduction of chemical modifications may cause undesired side effects, thereby impairing the gene regulatory efficacy. Herein, a methyl-engineered DNAzyme (MeDz) is developed for the visualization of endogenous alkyltransferase (AGT) and the simultaneous self-sufficiently on-demand gene regulation. The catalytic activity of DNAzyme can be efficiently blocked by O6-methylguanine (O6MeG) modification and specifically restored via the AGT-mediated DNA-repairing pathway. This simply designed MeDz is demonstrated to reveal AGT of varying expression levels in different cells, opening the possibility to explore the AGT-related biological processes. Moreover, the AGT-guided MeDz exhibits cell-selective regulation on the human early growth response-1 (EGR-1) gene, with efficient gene repression in breast cancer cells and low effectiveness in normal cells. The proposed MeDz offers an attractive strategy for on-demand gene regulation, displaying great potential in biomedical applications.

The Impact of Early Growth Response 1 (Egr1) on Hippocampal Synaptic Plasticity and Cognitive Function: Narrative Review

Introduction: EGR1 (Early Growth Response 1) gene expression is a molecular response that occurs in the brain as a result of synaptic activity and environmental stimuli. Early growth response 1 (EGR1) expression can be affected by several factors, including exercise or physical training. This review aims to determine the effect of EGR 1 expression on hippocampal synaptic plasticity function. Method: Literature search using data-based Pubmed, Science Direct, and Scopus online. The data used is from the year 1978 until the year 2022. Searched using English keywords such as EGR 1 and hippocampus. Results: Animal and human studies show that physical exercise can increase the expression of the EGR1 gene in the brain. This enhanced EGR1 expression is associated with increased synaptic plasticity, which includes changes in the strength and connectivity of synapses between neurons. Synaptic plasticity refers to the ability of the nervous system to change the strength and efficiency of communication between neurons. Physical exercise has been shown to increase synaptic plasticity by increasing dendritic growth and continuity, increasing neurogenesis (the formation of new neurons), and increasing synaptic connections between neurons. Physical exercise can increase EGR1 expression and synaptic plasticity. Increased EGR1 expression and synaptic plasticity induced by physical exercise are associated with improvements in cognitive functions, including memory, learning, and thinking ability. Conclusion: There is evidence that exercise can increase EGR1 expression and synaptic plasticity in the brain, especially in the hippocampus, to improve cognitive function.

EGR1 suppresses HCC growth and aerobic glycolysis by transcriptionally downregulating PFKL

BackgroundHepatocellular Carcinoma (HCC) is a matter of great global public health importance; however, its current therapeutic effectiveness is deemed inadequate, and the range of therapeutic targets is limited. The aim of this study was to identify early growth response 1 (EGR1) as a transcription factor target in HCC and to explore its role and assess the potential of gene therapy utilizing EGR1 for the management of HCC.MethodsIn this study, both in vitro and in vivo assays were employed to examine the impact of EGR1 on the growth of HCC. The mouse HCC model and human organoid assay were utilized to assess the potential of EGR1 as a gene therapy for HCC. Additionally, the molecular mechanism underlying the regulation of gene expression and the suppression of HCC growth by EGR1 was investigated.ResultsThe results of our investigation revealed a notable decrease in the expression of EGR1 in HCC. The decrease in EGR1 expression promoted the multiplication of HCC cells and the growth of xenografted tumors. On the other hand, the excessive expression of EGR1 hindered the proliferation of HCC cells and repressed the development of xenografted tumors. Furthermore, the efficacy of EGR1 gene therapy was validated using in vivo mouse HCC models and in vitro human hepatoma organoid models, thereby providing additional substantiation for the anti-cancer role of EGR1 in HCC. The mechanistic analysis demonstrated that EGR1 interacted with the promoter region of phosphofructokinase-1, liver type (PFKL), leading to the repression of PFKL gene expression and consequent inhibition of PFKL-mediated aerobic glycolysis. Moreover, the sensitivity of HCC cells and xenografted tumors to sorafenib was found to be increased by EGR1.ConclusionOur findings suggest that EGR1 possesses therapeutic potential as a tumor suppressor gene in HCC, and that EGR1 gene therapy may offer benefits for HCC patients.

Protection of Chickens from Mycoplasma gallisepticum through the MAPK/ERK/JNK Pathway by a Compound of Ten Chinese Medicine Formulas1

Mycoplasma gallisepticum (MG) is a common avian pathogen that mainly infects poultry, causing significant reductions in body weight gain and egg production, along with damage to immune organs and immunosuppression. MG is susceptible to co-infections with other pathogens, leading to increased mortality rates and significant economic losses in the global poultry industry. While antibiotics have been extensively applied worldwide to treat MG infections in poultry production, concerns regarding antibiotic resistance and residue remain prevalent. Traditional Chinese medicine (TCM), renowned for its natural, safe, and non-toxic properties, has shown significant anti-inflammatory and immune-enhancing effects. This study aimed to investigate the protective effect of TCM on production performance and its impact on MG-induced immunosuppression through the MAPK/ERK/JNK signaling pathway in chickens. Our results showed that TCM alleviated the negative effects of MG infection on production performance, as evidenced by improvements in body weight gain, feed conversion rate, survival rate, and immune organ index. TCM exhibited direct inhibition of the MG proliferation in vitro and in vivo. Furthermore, TCM treatment promoted the normalization of trachea and lung tissue structure in MG-infected chickens, leading to a significant reduction in inflammatory damage. Moreover, following the treatment with the TCM, the production of pro-inflammatory cytokines Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) decreased significantly, accompanied by the downregulation of pro-apoptotic genes caspase3, caspase9, and BAX, both in vitro and in vivo. A mechanism-based study showed that in vitro and in vivo treatment with the TCM significantly reduced the expression of key proteins, including early growth response gene1 (EGR1), p-ERK, p-JNK, and p-JUN. Altogether, TCM improved body weight gain, inhibited pro-inflammation responses, and alleviated tissue damage by inhibiting the MAPK/ERK/JNK signaling pathway to protect the performance and immune system of MG-infected chickens.

EGR1 functions as a new host restriction factor for SARS-CoV-2 to inhibit virus replication through the E3 ubiquitin ligase MARCH8.

Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma

AbstractThe use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell–like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

Systems assessment of statins hazard: Integrating in silico prediction, developmental toxicity profile and transcriptomics in zebrafish

Statins are prescribed widely as lipid-lowering agents. However, statins are associated with an increased harmful risk on public health and the ecosystem. Little is known about statins’ toxicity on biological development and the underlying molecular mechanisms. We exposed zebrafish embryos to a series of statins to evaluate their development toxicity. Statins-induced embryonic developmental defects in a concentration-dependent manner. 72 h LC50 values for lovastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin, and pravastatin were 0.01 μM, 0.04 μM, 1.93 μM, 37.28 μM, 79.29 μM, and 2170 μM, respectively. Moreover, the expression of genes involved in heart contraction, calcium ion binding, transcription factors, nucleus, and G protein-coupled receptor signaling pathway was altered by statins. The early growth response gene (egr4) and transcription factor genes (fosab and fosb) were screened as potential toxicity targets due to their significant upregulation based on protein-protein interaction (PPI) and drug-gene interaction network analysis. Finally, the ecotoxicity profile of statins was predicted by in silico method, and statins were high or moderate risk to aquatic organisms. We provide a systems toxicology strategy to explore the toxicity of statins and illustrate the potential mechanisms of action.

The Landscape of Early Growth Response Family Members 1-4 in Hepatocellular Carcinoma: Their Biological Roles and Diagnostic Utility.

The incidence of hepatocellular carcinoma (HCC), which is one of the most frequent types of cancer seen all over the world, is steadily growing from year to year. EGR genes are members of the early growth response (EGR) gene family. It has been shown that EGR genes play an increasingly essential role in the development of tumors and the progression of numerous malignancies. However, the possible diagnostic and prognostic roles of EGR genes in HCC have only been examined in a limited number of studies. Expression and methylation data on EGR family members were obtained from TCGA datasets. The prognostic values of EGR members were studied. Additionally, the correlations of EGR members with immune cells were assessed through the single-sample gene set enrichment analysis (ssGSEA). In this study, we found that the expression of EGR1, EGR2, EGR3, and EGR4 was distinctly decreased in HCC specimens compared with nontumor specimens. ROC assays confirmed that they have a strong ability in screening HCC specimens from nontumor specimens. According to the findings of Pearson's correlation, EGR1, EGR2, EGR3, and EGR4 were found to have a negative association with the methylation level. Survival study revealed that EGR1, EGR2, and EGR3 were associated with the clinical outcome of HCC patients. Immune cell enrichment analysis demonstrated that the expressions of all EGR members were positively related to the levels of most types of immune cells, such as macrophages, NK cells, B cells, T cells, eosinophils, and CD8 T cells. Overall, the current work demonstrated the expression mode and prognostic value of EGR members in HCC in a comprehensive manner, offering insights for further research of the EGR family as possible clinical biomarkers in HCC.

Abstract 2777: Characterizing the molecular underpinnings of sorafenib sensitivity in desmoid tumors

Abstract Background: Desmoid tumors (DT) are rare fibroblastic sarcomas that have high recurrence rates. Though we know mutations in the beta-catenin (CTNNB1) gene are commonly observed with dysregulation of the Wnt signaling pathway in patients with DT, the molecular mechanisms driving DT growth remain poorly understood. There is currently no standard systemic treatment for DT. The multi-kinase inhibitor, sorafenib, has been identified as a potential front-line agent for DT after a recent clinical trial demonstrated an objective response in one third of patients. The mechanism for how sorafenib inhibits DT progression is still unclear, so it is unknown which characteristics predict response to the drug. Our work has identified the early growth response-1 (EGR1) gene as a downstream target of sorafenib. Methods: Nine DT cell lines have previously been brought into culture with stable passage. These cell lines were treated with sorafenib at 10 μM. RNA was collected from parental and treated cells and subsequent RNA sequencing was performed. Differential gene expression was performed using R. Appropriate comparisons for multiplicity were performed. Western blots were performed to confirm changes in expression of key genes at the protein level. EGR1 overexpression vector was introduced to the 2 DT cell lines and further treated with sorafenib. Additional banked patient desmoid tumor samples were sequenced to assess EGR1 levels and correlation with the CTNNB1. Results: We first evaluated the effect of sorafenib on CTNNB1 in the DT models. Western blot demonstrated no changes in protein levels of CTNNB1 in the 4 cell lines tested at 72 hours post-treatment. This was further confirmed on RNA-sequencing. To identify additional gene targets of sorafenib, additional differential gene expression analysis was performed resulting in 13 altered genes at an adjusted p < 0.01. The most significantly altered gene, both by p-value and by fold change, was the EGR1 mRNA transcript. EGR1 transcript levels were significantly attenuated by sorafenib treatment (logFC = 2.720, adjusted p-value << 0.0001). Western blot analysis confirmed this decrease. EGR1-high DT cell line models exhibited faster growth rates and also higher sensitivity to sorafenib. Fifteen additional patient desmoid tumor samples confirmed the steady state of CTNNB1 and high variability of the EGR1 transcript. There was no correlation between the EGR1 and CTNNB1 transcript. Conclusion: This study found that sorafenib does not directly modulate CTNNB1, but does decrease EGR1 expression at both the mRNA and protein level. This suggests that EGR1 expression may be associated with aggressive DT and may also predict responsiveness to treatment with sorafenib. Confirmatory EGR1 modulation experiments are underway and will be provided at time of presentation. Citation Format: Max Devine, Joy Tang, Colin Stets, John Hays, James L. Chen. Characterizing the molecular underpinnings of sorafenib sensitivity in desmoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2777.

Understanding pathogen-host interplay by expression profiles of lncRNA and mRNA in the liver of Echinococcus multilocularis-infected mice.

Almost all Echinococcus multilocularis (Em) infections occur in the liver of the intermediate host, causing a lethal zoonotic helminthic disease, alveolar echinococcosis (AE). However, the long non-coding RNAs (lncRNAs) expression profiles of the host and the potential regulatory function of lncRNA during Em infection are poorly understood. In this study, the profiles of lncRNAs and mRNAs in the liver of mice at different time points after Em infection were explored by microarray. Thirty-one differentially expressed mRNAs (DEMs) and 68 differentially expressed lncRNAs (DELs) were found continuously dysregulated. These DEMs were notably enriched in “antigen processing and presentation”, “Th1 and Th2 cell differentiation” and “Th17 cell differentiation” pathways. The potential predicted function of DELs revealed that most DELs might influence Th17 cell differentiation and TGF-β/Smad pathway of host by trans-regulating SMAD3, STAT1, and early growth response (EGR) genes. At 30 days post-infection (dpi), up-regulated DEMs were enriched in Toll-like and RIG-I-like receptor signaling pathways, which were validated by qRT-PCR, Western blotting and downstream cytokines detection. Furthermore, flow cytometric analysis and serum levels of the corresponding cytokines confirmed the changes in cell-mediated immunity in host during Em infection that showed Th1 and Th17-type CD4+ T-cells were predominant at the early infection stage whereas Th2-type CD4+ T-cells were significantly higher at the middle/late stage. Collectively, our study revealed the potential regulatory functions of lncRNAs in modulating host Th cell subsets and provide novel clues in understanding the influence of Em infection on host innate and adaptive immune response.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Hyperoxia/Hypoxia Exposure Primes a Sustained Pro-Inflammatory Profile of Preterm Infant Macrophages Upon LPS Stimulation.

Preterm infants are highly susceptible to sustained lung inflammation, which may be triggered by exposure to multiple environmental cues such as supplemental oxygen (O2) and infections. We hypothesized that dysregulated macrophage (MФ) activation is a key feature leading to inflammation-mediated development of bronchopulmonary dysplasia (BPD) in preterm infants. Therefore, we aimed to determine age-dependent differences in immune responses of monocyte-derived MФ comparing cord blood samples derived from preterm (n=14) and term (n=19) infants as well as peripheral blood samples from healthy adults (n=17) after lipopolysaccharide (LPS) exposure. Compared to term and adult MФ, LPS-stimulated preterm MФ showed an enhanced and sustained pro-inflammatory immune response determined by transcriptome analysis, cytokine release inducing a RORC upregulation due to T cell polarization of neonatal T cells, and TLR4 surface expression. In addition, a double-hit model was developed to study pulmonary relevant exposure factors by priming MФ with hyperoxia (O2 = 65%) or hypoxia (O2 = 3%) followed by lipopolysaccharide (LPS, 100ng/ml). When primed by 65% O2, subsequent LPS stimulation in preterm MФ led to an exaggerated pro-inflammatory response (e.g. increased HLA-DR expression and cytokine release) compared to LPS stimulation alone. Both, exposure to 65% or 3% O2 together with subsequent LPS stimulation, resulted in an exaggerated pro-inflammatory response of preterm MФ determined by transcriptome analysis. Downregulation of two major transcriptional factors, early growth response gene (Egr)-2 and growth factor independence 1 (Gfi1), were identified to play a role in the exaggerated pro-inflammatory response of preterm MФ to LPS insult after priming with 65% or 3% O2. Preterm MФ responses to LPS and hyperoxia/hypoxia suggest their involvement in excessive inflammation due to age-dependent differences, potentially mediated by downregulation of Egr2 and Gfi1 in the developing lung.

CARD19, a Novel Negative Regulator of B-Cell Tolerance

The CARD11-Bcl10-Malt1 (CBM) signalosome controls TAK1 activation to regulate B-cell receptor (BCR)-induced NF-κB activation and B cell biology. The biological function of caspase recruitment domain family member 19 (CARD19), originally identified as a BCL10-interacting CARD protein (BinCARD), is not known. Here we found CARD19 strongly interacted with TAK1 but not BCL10 or other CBM components and prevented TAK1's association with TAB2, thereby inhibiting TAB2-mediated TAK1 ubiquitination and activation and subsequent NF-κB activation. CARD19 was ubiquitously expressed in hematopoietic lineages but its deficiency in mice had no effect on hematopoiesis, including B cell development and humoral immune response. CARD19 deficiency enhanced clonal deletion, receptor editing and anergy of self-reactive B cells, thus reducing autoantibody production in vivo. Mechanistically, CARD19 deficiency led to an increase of BCR/TAK1-mediated NF-κB activation. Activation of NF-κB, such as c-Rel, was responsible for the up-regulation of BCR-induced expression of the transcription factor early growth response genes 2 and 3 (Egr2, Egr3) and the E3 ubiquitin ligases, c-Cbl and Cbl-b, the important inducers of B-cell tolerance in B cells. Further, high-throughput RNA sequencing analysis revealed that CARD19 deficiency did not affect the overall antigen-induced gene expression in naïve B cells but suppressed BCR signaling to increase hyporesponsiveness of self-reactive B cells. Consequently, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus (SLE) and autoimmunity in a B cell-intrinsic manner. Taken together, CARD19 negatively regulates BCR-induced NF-κB activation via blocking TAK1/TAB2 interaction and its deficiency leads to NF-κB-induced expression of Egr2/3 and c-Cbl/Cbl-b in self-reactive B cells, which enhances B-cell tolerance and thus prevents autoimmunity. DisclosuresNo relevant conflicts of interest to declare.

Betulinic acid promotes the osteogenic differentiation of human periodontal ligament stem cells by upregulating EGR1

Periodontitis is one of the most common chronic inflammations of the oral cavity, which eventually leads to tooth loss. Betulinic acid (BetA) is an organic acid that has anti-inflammatory effects and is derived from fruits and plants, but its effect on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is still unclear. This study aimed to explore the effect of BetA on the osteogenic differentiation of hPDLSCs and its mechanism. Our results revealed that BetA not only promoted the viability of hPDLSCs but also induced their osteogenic differentiation in a dose-dependent manner. In addition, RNA sequencing was used to screen the differentially expressed genes (DEGs) after hPDLSCs were treated with BetA, and 127 upregulated and 138 downregulated genes were identified. Gene Ontology enrichment analysis showed that DEGs were mainly involved in the response to lithium ions and the positive regulation of macrophage-derived foam cell differentiation. The Kyoto Encyclopedia of Genes and Genomes analysis results revealed that DEGs were enriched in the nuclear factor-κB and interleukin-17 signaling pathways. More importantly, we confirmed that early growth response gene 1 (EGR1), one of the three DEGs involved in bone formation, significantly promoted the expression of osteogenic markers and the mineralization of hPDLSCs. Knockdown of EGR1 obviously limited the effect of BetA on the osteogenic differentiation of hPDLSCs. In conclusion, BetA promoted the osteogenic differentiation of hPDLSCs through upregulating EGR1, and BetA might be a promising candidate in the clinical application of periodontal tissue regeneration.

Detecting Abnormal Neuronal Activity in a Chronic Migraine Model by Egr1-EGFP Transgenic Mice

Chronic migraine (CM) is a highly disabling neurological disorder characterized by recurrent headache accompanied by a variety of sensory and/or emotional symptoms. However, the mechanisms of migraine onset and its chronicity have not been elucidated. The present study was designed to search for brain regions and neurons that were abnormally activated by CM and might be related to its pathogenesis and different concomitant symptoms. CM models were established here by repeated intraperitoneal injection of nitroglycerin (NTG) every other day for 9 days to early growth response gene 1 (Egr1)-enhanced green fluorescent protein (EGFP) transgenic mice, which allowed monitoring of neuronal activities in the whole brain. CM-related behaviors were recorded through head grooming test and light aversion assay. Elevation of Egr1 expression signals was detected in trigeminal nucleus caudalis (TNC), primary somatosensory cortex (SSp), lateral amygdala nucleus (LA), primary visual area (VISp), and temporal association areas (TEa) 2 h after the last injection of NTG by immunofluorescence and digital slice scanning technology. Meanwhile, no change of Egr1 expression was found in auditory areas (AUD), CA1, ectorhinal area (ECT), piriform (PIR), and anterior cingulate area (ACC). Furthermore, with the strongest support by evidence-based medicine among the current limited oral treatments of CM, topiramate was administrated every day for 11 days from 2 days before the first NTG injection. The results showed that topiramate partially improved the photophobia behavior of CM models in the short-term with gradually weakened efficacy as the course of the disease prolonged. Meanwhile, NTG-induced increase in Egr1 expression was completely reversed in TNC, SSp, and VISp and partially reduced in LA and TEa by topiramate at the same time point mentioned above. In conclusion, the current results suggested that the abnormal hyperactivities in TNC, SSp and VISp were associated with the pathogenesis of CM.

Phragmunis a suppresses glioblastoma through the regulation of MCL1-FBXW7 by blocking ELK1-SRF complex-dependent transcription

Glioblastoma (GBM) is a highly aggressive brain tumor. During screening work, we found a new compound named phragmunis A (PGA), which is derived from the fruitbody of Trogia venenata, exhibits a potential cytotoxic effect on patient-derived recurrent GBM cells and temozolomide (TMZ)-resistant cell lines. The present study was designed to investigate the potential molecular mechanism of the anti-glioma effects of PGA in vitro and in vivo. Studies investigating the mechanism revealed that PGA diminished the binding efficiency of ETS family of transcription factor (ELK1) and Serum response factor (SRF), and suppressed ELK1-SRF complex-dependent transcription, which decreased the transcriptional levels of downstream genes Early growth response protein 1 (EGR1)-Polycomb ring finger (BMI1), thus inducing the imbalanced regulation between Myeloid cell leukaemia-1 (MCL1) and F-Box and WD repeat domain containing 7 (FBXW7). Finally, orthotopic xenograft models were established to confirm the anti-glioma effect of PGA on tumour growth. We showed, for the first time, that the cytotoxic effects of PGA occurred by inducing MCL1 inhibition and FBXW7 activation by blocking ELK1-SRF complex-dependent transcription. The blockage of ELK1-mediated transcription resulted in the suppression of EGR1-BMI1, which led to the upregulation of FBXW7 expression and downregulation of MCL1. These findings suggested that PGA could be a therapeutic drug candidate for the treatment of recurrent GBM by targeting the ELK1-SRF complex.

EGR2 plays a differential immune regulatory role in normal C57BL/6 mice and autoimmune-prone B6/lpr mice

Abstract Early growth response protein 2 (EGR2) is a zinc-finger transcription factor that belongs to the early growth response (Egr) gene family. Recent studies have shown that EGR2 is required to induce T cell anergy, and it plays an important role in T cell immunity and autoimmunity. Conditional depletion of EGR2 in T lymphocytes in C57BL/6 (B6) mice (EGR2−/−B6) was able to break immune tolerance and induce T cell-driven, a lupus-like autoimmune disease. In EGR2−/−B6 mice, there was also enhanced GC response and humoral responses. However, increased EGR2 expression has been observed in lupus and autoimmune disorder systemic sclerosis. These observations seem to contradict the autoimmune suppressive role of EGR2 in B6 mice. To investigate further the immune regulatory role of EGR2 in an autoimmune context, we derived EGR2−/−B6/lpr mice with EGR2 deficiency in both T and B cells. We found that EGR2 depletion promoted splenomegaly and increased IFNγ, IL-10, IL-2 in both B6 and B6/lpr mice. Significantly, we found that in B6/lpr mice, EGR2 depletion reduced serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. Furthermore, in B6/lpr mice, depletion of EGR2 promoted germinal center B (GCB) cell development, but suppressed plasma cell (PC) differentiation. However, in wild-type B6 mice, EGR2 depletion significantly increased anti-dsDNA autoantibodies, accompanied by a significant increase of GCB cell development and a trend of increase in PC differentiation. Together, we demonstrated that EGR2 has a differential role in the normal and autoimmune state, particularly in regulating GCB transition into plasma cell during late B cell development.

Pharmacological profiles of 5‐HT 2A Receptor (5‐HT 2A R):5‐HT 2C R Interactions In Vitro

Aims The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR have garnered intense interest from drug discovery programs due to their role in metabolic (e.g., obesity) and neuropsychiatric disorders (e.g., addiction, depression, psychosis). Functional and physical interactions between the 5-HT2AR and 5-HT2CR occur in living cells (Felsing et al., PLoS One 13(8):e0203137, 2018) and rat brain (Price et al., ACS Chem Neurosci 10:3241, 2019). Intriguingly, when the two receptors are in complex, the 5-HT2CR is functionally dominant, i.e., silences signaling from the 5-HT2AR in vitro (Moutkine et al., J Biol Chem 292:6352, 2017). In the present study, we have extended these observations to uncover additional cellular signaling output associated with the 5-HT2AR:5-HT2CR complex and explore disruption of the receptor interfaces important for their interaction and signaling. Methods . A split luciferase complementation assay (LCA) was employed to assess the direct coupling of 5-HT2AR and 5-HT2CR in live HEK293 cells. The cDNA for the human 5-HT2AR and 5-HT2CR fused with complementary N-terminal (NLuc) and C-terminal (CLuc) inactive fragments, respectively, were transfected into HEK293 cells using template mammalian expression vectors and standard cloning techniques. Association of the receptor constructs in vitro within ~50 nm reconstitutes luciferase activity and light is released in the presence of D-luciferin. The expression of the activity-regulated immediate early gene early growth response 1 (EGR1) was used to trace cellular responsivity to 5-HT in cells. A truncated version of the 5-HT2CR transmembrane helix IV (TM4) domain conjugated to a polyethylene glycol chain (PEG-TM4 5-HT2CR) was synthesized as a tool to disrupt formation of the 5-HT2AR:5-HT2CR complex. Results . Serotonin (5-HT) readily induced robust expression of EGR1 mRNA in cells solely expressing 5-HT2AR, but not 5-HT2CR; 5-HT-evoked EGR1 expression was blunted in cells co-expressing the 5-HT2CR plus 5-HT2AR. In the LCA, vehicle (0.1% DMSO) treated cells exhibit robust complementation (i.e., increased luminescence) between the 5-HT2AR and 5-HT2CR. The PEG-TM4 5-HT2CR peptide significantly decreased complementation (p<0.05) in a concentration-dependent manner indicating an inhibition of association between the 5-HT2AR and 5-HT2CR. Conclusions Blunted 5-HT-evoked EGR1 expression in cells expressing both receptors supports the “master regulator” role for the 5-HT2CR when in complex with the 5-HT2AR. Secondly, the formation of the 5-HT2AR:5-HT2CR complex in vitro is dependent on an interaction interface located at the 5-HT2CR TM4. Ongoing studies are testing the hypothesis that the PEG-TM4 5-HT2CR peptide restores a critical signaling output of 5-HT2AR function, which will indicate the importance of allosteric regulation by the heteromer complex. Future studies with our novel TM domain peptides will help elucidate determinants of receptor complex formation and signaling and the physiological relevance of the 5-HT2AR:5-HT2CR complex in vivo.

Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis.

Long non-coding RNA (lncRNA) myocardial infarction-associated transcript (MIAT) has been widely-demonstrated to function as diagnostic markers for acute myocardial infarction (MI). This study was designed to explore the modulatory role of MIAT and its underlying molecular mechanism in MI. Firstly, MI mouse model was developed via ligation of the descending branch of the left coronary artery, and cell model was established through exposure to hypoxic conditions. Online prediction indicated that MIAT could bind to microRNA-10a-5p (miR-10a-5p), while miR-10a-5p was highlighted to bind to early growth response gene-2 (EGR2). MIAT and EGR2 were subsequently determined to be highly-expressed, whereas miR-10a-5p was found to be poorly-expressed in cardiomyocytes exposed to hypoxia as well as in MI mice using RT-qPCR and Western blot assay. The binding relationships between MIAT and miR-10a-5p, and between miR-10a-5p and EGR2 were further confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. The results of in vitro and in vivo experimentation also suggested that overexpression of miR-10a-5p or silencing of MIAT and EGR2 reduced cardiomyocyte apoptosis and increased ATP content, thus alleviating the impairment of cardiac function following MI. In a word, inhibition of MIAT protects against cardiac dysfunction induced by MI through the crosstalk with miR-10a-5p/EGR2.