Egr-1 Knockout Mice Research Articles

Early growth response 1 exacerbates thoracic aortic aneurysm and dissection of mice by inducing the phenotypic switching of vascular smooth muscle cell through the activation of Krüppel-like factor 5.

Vascular smooth muscle cell (VSMC) phenotypic switching has been reported to regulate vascular function and thoracic aortic aneurysm and dissection (TAAD) progression. Early growth response 1 (Egr1) is associated with the differentiation of VSMCs. However, the mechanisms through which Egr1 participates in the regulation of VSMCs and progression of TAAD remain unknown. This study aimed to investigate the role of Egr1 in the phenotypic switching of VSMCs and the development of TAAD. Wild-type C57BL/6 and SMC-specific Egr1-knockout mice were used as experimental subjects and fed β-aminopropionitrile for 4 weeks to construct the TAAD model. Ultrasound and aortic staining were performed to examine the pathological features in thoracic aortic tissues. Transwell, wound healing, CCK8, and immunofluorescence assays detected the migration and proliferation of synthetic VSMCs. Egr1 was directly bound to the promoter of Krüppel-like factor 5 (KLF5) and promoted the expression of KLF5, which was validated by JASPAR database and dual-luciferase reporter assay. Egr1 expression increased and was partially co-located with VSMCs in aortic tissues of mice with TAAD. SMC-specific Egr1 deficiency alleviated TAAD and inhibited the phenotypic switching of VSMC. Egr1 knockdown prevented the phenotypic switching of VSMCs and subsequently suppressed the migration and proliferation of synthetic VSMCs. The inhibitory effects of Egr1 deficiency on VSMCs were blunted once KLF5 was overexpressed. Egr1 aggravated the development of TAAD by promoting the phenotypic switching of VSMCs via enhancing the transcriptional activation of KLF5. These results suggest that inhibition of SMC-specific Egr1 expression is a promising therapy for TAAD.

Early Growth Response Protein 1 Exacerbates Murine Inflammatory Bowel Disease by Transcriptional Activation of Matrix Metalloproteinase 12.

Inflammatory bowel disease (IBD) is an inflammatory condition affecting the colon and small intestine, with Crohn's disease and ulcerative colitis being the major types. Individuals with long-term IBD are at an increased risk of developing colorectal cancer. Early growth response protein 1 (Egr1) is a nuclear protein that functions as a transcriptional regulator. Egr1 is known to control the expression of numerous genes and play a role in cell growth, proliferation, and differentiation. While IBD has been associated with severe inflammation, the precise mechanisms underlying its pathogenesis remain unclear. This study aimed to investigate the role of Egr1 in the development of IBD. High levels of Egr1 expression were observed in a mouse model of colitis induced by dextran sulfate sodium (DSS), as determined by immunohistochemical (IHC) staining. Chronic DSS treatment showed that Egr1 knockout (KO) mice exhibited resistance to the development of IBD, as determined by changes in their body weight and disease scores. Additionally, enzyme-linked immunosorbent assay (ELISA) and IHC staining demonstrated decreased expression levels of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as matrix metalloproteinase 12 (MMP12). Putative Egr1 binding sites were identified within the MMP12 promoter region. Through reporter assays and chromatin immunoprecipitation (ChIP) analysis, it was shown that Egr1 binds to the MMP12 promoter and regulates MMP12 expression. In conclusion, we found that Egr1 plays a role in the inflammation process of IBD through transcriptionally activating MMP12.

Sex-Linked Growth Disorder and Aberrant Pituitary Gene Expression in Nestin-Cre-Mediated Egr1 Conditional Knockout Mice.

Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.

EGR1 is crucial for the chlorogenic acid-provided promotion on liver regeneration and repair after APAP-induced liver injury.

Improper use of acetaminophen (APAP) will induce acute liver failure. This study is designed to investigate whether early growth response-1 (EGR1) participated in the promotion on liver repair and regeneration after APAP-induced hepatotoxicity provided by natural compound chlorogenic acid (CGA). APAP induced the nuclear accumulation of EGR1 in hepatocytes regulated by extracellular-regulated protein kinase (ERK)1/2. In Egr1 knockout (KO) mice, the liver damage caused by APAP (300 mg/kg) was more severe than in wild-type (WT) mice. Results of chromatin immunoprecipitation and sequencing (ChIP-Seq) manifested that EGR1 could bind to the promoter region in Becn1, Ccnd1, and Sqstm1 (p62) or the catalytic/modify subunit of glutamate-cysteine ligase (Gclc/Gclm). Autophagy formation and APAP-cysteine adduct (APAP-CYS) clearance were decreased in Egr1 KO mice administered with APAP. The EGR1 deletion reduced hepatic cyclin D1 expression at 6, 12, or 18 h post APAP administration. Meanwhile, the EGR1 deletion also decreased hepatic p62, Gclc and Gclm expression, GCL enzymatic activity, and glutathione (GSH) content and decreased nuclear factor erythroid 2-related factor 2 (Nrf2) activation and thus aggravated oxidative liver injury induced by APAP. CGA increased EGR1 nuclear accumulation; enhanced hepatic Ccnd1, p62, Gclc, and Gclm expression; and accelerated the liver regeneration and repair in APAP-intoxicated mice. In conclusion, EGR1 deficiency aggravated liver injury and obviously delayed liver regeneration post APAP-induced hepatotoxicity through inhibiting autophagy, enhancing liver oxidative injury, and retarding cell cycle progression, but CGA promoted the liver regeneration and repair in APAP-intoxicated mice via inducing EGR1 transcriptional activation.

HMGB1 induced endothelial to mesenchymal transition in liver fibrosis: The key regulation of early growth response factor 1

BackgroundLiver fibrosis has been the focus and difficulty of medical research in the world and its concrete pathogenesis remains unclear. This study aims to observe the high-mobility group box 1 (HMGB1)-induced hepatic endothelial to mesenchymal transition (EndoMT) during the development of hepatic fibrosis, and further to explore the crucial involvement of Egr1 in this process. MethodsCarbon tetrachloride (CCl4), diosbulbin B (DB), N-acetyl-p-aminophenol (APAP) and bile duct ligation (BDL) were used to induce liver fibrosis in mice. Serum HMGB1 content, the occurrence of EndoMT and the production of extracellular matrix (ECM) in vitro and in vivo were detected by Western-blot. ResultsThe elevated serum HMGB1 content, the occurrence of EndoMT, the production of ECM and the activation of Egr1 were observed in mice with liver fibrosis induced by CCl4, DB, APAP or BDL. HMGB1 induced EndoMT and ECM production in human hepatic sinusoidal endothelial cells (HHSECs), and then HHSECs lost the ability to inhibit the activation of hepatic stellate cells (HSCs). The hepatic deposition of collagen, the increased serum HMGB1 content and hepatic EndoMT were further aggravated in Egr1 knockout mice. Natural compound silymarin attenuated liver fibrosis in mice induced by CCl4 via increasing Egr1 nuclear accumulation, decreasing serum HMGB1 content and inhibiting hepatic EndoMT. ConclusionEgr1 regulated the expression of HMGB1 that induced hepatic EndoMT, which plays an important role in the development of liver fibrosis.General significance:This study provides a novel therapeutic strategy for the treatment of liver fibrosis in clinic.

Egr1 confers protection against acetaminophen‑induced hepatotoxicity via transcriptional upregulating of Acaa2.

Background: Acetaminophen (APAP)-induced liver injury (AILI) is a common cause of drug-induced liver injury (DILI). The mechanism underlying protection in AILI or DILI remains to be elucidated, and the role of early growth response 1 (Egr1) in AILI and potential mechanisms remain to be known.Methods: The role of Egr1 was studied both in vivo and in vitro. Liver-specific Egr1-knockout (Egr1LKO) mice and those overexpressing Egr1 via tail vein injection of Egr1-expressing adenovirus (Ad-Egr1) were utilized with AILI. Chromatin immunoprecipitation-sequencing, RNA-sequencing, seahorse XF analysis, and targeted fatty acid analysis were performed. EGR1 levels were also studied in liver tissues and serum samples from AILI/DILI patients.Results: In this study, we have demonstrated that Egr1 was upregulated in AILI models in vivo and in vitro. liver-specific Egr1 knockout aggravated AILI; however, Ad-Egr1 treatment ameliorated this. Mechanistically, Egr1 deficiency inhibited, whereas overexpression promoted, mitochondrial respiratory function and fatty acid β-oxidation (FAO) activity in AILI. Egr1 transcriptionally upregulated FAO-related genes in hepatocytes. Notably, the knockdown of acetyl-coenzyme A acyltransferase 2 (Acaa2), a key gene involved in FAO, diminished this protective effect of Egr1. Clinically, EGR1 was markedly increased in liver tissues from AILI patients. Interestingly, EGR1 levels of liver tissues and serum samples were also obviously higher in idiosyncratic DILI patients.Conclusions: Egr1 confers adaptive protection in AILI, mediated via the transcriptional upregulation of Acaa2, which improves mitochondrial FAO, and might be a potential biomarker and novel therapeutic target for AILI.

EGR-1 acts as a transcriptional activator of KLK7 under IL-13 stimulation

Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine peptidase that plays a crucial role in regulating skin desquamation. KLK7 expression is highly upregulated in atopic dermatitis (AD) skin lesions in both humans and mice. Th2-lymphocyte-derived cytokines, including interleukin (IL)-4 and IL-13, have been shown to promote KLK7 expression in keratinocytes in patients with AD. However, the molecular mechanism underlying KLK7 expression remains poorly understood. Here, we demonstrated that the EGR-1-binding sequence (EBS) in the promoter region of KLK7 played a crucial role in IL-13-induced KLK7 transcription. Disruption of the EBS induced by a point mutation inhibited IL-13-induced KLK7 promoter activity. EGR-1 was shown to directly bind to the EBS, and EGR1 knockdown with shRNA abrogated IL-13-induced KLK7 expression. Using Egr1 knockout mice, we showed that Egr-1 was necessary for KLK7 expression in AD-like lesions induced by the repeated topical application of 2,4-dinitrobenzene on the dorsal skin of mice. We also demonstrated that the ERK1/2 mitogen-activated protein kinase (MAPK) pathway was responsible for EGR-1-dependent KLK7 transcription in response to IL-13 stimulation. Our findings delineate a signaling pathway that contributes to the regulation of KLK7 expression through the IL13-ERK MAPK-EGR1 signaling axis.

Epigenetic regulation of neuronal cell specification inferred with single cell “Omics” data

The brain is a highly complex organ consisting of numerous types of cells with ample diversity at the epigenetic level to achieve distinct gene expression profiles. During neuronal cell specification, transcription factors (TFs) form regulatory modules with chromatin remodeling proteins to initiate the cascade of epigenetic changes. Currently, little is known about brain epigenetic regulatory modules and how they regulate gene expression in a cell-type specific manner. To infer TFs involved in neuronal specification, we applied a recursive motif search approach on the differentially methylated regions identified from single-cell methylomes. The epigenetic transcription regulatory modules (ETRM), including EGR1 and MEF2C, were predicted and the co-expression of TFs in ETRMs were examined with RNA-seq data from single or sorted brain cells using a conditional probability matrix. Lastly, computational predications were validated with EGR1 ChIP-seq data. In addition, methylome and RNA-seq data generated from Egr1 knockout mice supported the essential role of EGR1 in brain epigenome programming, in particular for excitatory neurons. In summary, we demonstrated that brain single cell methylome and RNA-seq data can be integrated to gain a better understanding of how ETRMs control cell specification. The analytical pipeline implemented in this study is freely accessible in the Github repository (https://github.com/Gavin-Yinld/brain_TF).

Hepatocyte Early Growth Response 1 (EGR1) Regulates Lipid Metabolism in Nonalcoholic Fatty Liver Disease

ObjectiveNonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide due to significant increases in the prevalence of obesity, insulin resistance diabetes, and hyperlipidemia. A risk factor for NAFLD is insulin resistance as it promotes the excessive accumulation of lipids into the hepatocytes, causing cellular stress and death. As a stress response gene, early growth response 1 (EGR1) plays critical roles in inflammation and tissue repair. The aim for this study is to explore a novel role of hepatocyte EGR1 in the regulation of lipid metabolism in NAFLD.MethodsHuman microarray data from NCBI GEO database (accession #GSE48452) was used to obtain relative EGR1 expression in human normal and NAFLD livers. To determine the expression of EGR1 during the development of NAFLD, C57Bl6/J mice were fed a high in fat, cholesterol, and fructose (HFCF) for up to 9 months and EGR1 levels were examined. An in vitro insulin stimulation experiment was performed in wild type and Egr1‐deficient primary hepatocytes to assess insulin response. In vivo hepatocyte‐specific Egr1 knockout (Egr1‐HKO) mice was accomplished in 2‐month‐old Egr1flox/flox male mice by tail vein administration of adeno‐associated viral vector 8 (AAV8) expressing cre recombinase driven by thyroxine binding globulin (TBG) promoter (AAV8‐Tbg‐Cre). An empty vector AAV8‐Tbg‐Null was included as a control. WT and Egr1‐HKO mice were fed either a normal chow or HFCF diet for 5 months and followed by the examination of body composition, adiposity, metabolic rates, insulin sensitivity, and liver histology.ResultsEGR1 was dramatically induced by insulin and abundantly expressed in the postprandial liver. The expression of EGR1 was significantly reduced in human and mouse NAFLD livers, suggesting a biological relevance of EGR1 downregulation during the pathogenesis of NAFLD. In vitro hepatocyte cultures showed that Egr1‐deficient hepatocytes have impaired activation of AKT but enhanced de novo lipogenesis in response to insulin. In vivo knockout of hepatocyte‐specific Egr1 led to liver steatosis and increased whole body adiposity under the chow diet. Additionally, in vivo EGR1 deficiency in hepatocytes exacerbated NAFLD progression by promoting liver inflammation and fibrosis in mice fed a HFCF diet.ConclusionsOur studies reveal a previously unrecognized role of hepatic EGR1 in the regulation of lipid metabolism. Our findings suggest that hepatocyte EGR1 is crucial to maintain hepatic insulin response and loss of EGR1 in hepatocytes leads to liver steatosis that exacerbates NAFLD development and progression.Support or Funding InformationNCI K22CA184146 and NIH P20 GM103549 to Yuxia Zhang.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Egr-1 is required for neu/HER2-induced mammary tumors

Egr-1 is known to function mainly as a tumor suppressor through direct regulation of multiple tumor suppressor genes. To determine the role of Egr-1 in breast tumors in vivo, we used mouse models of breast cancer induced by HER2/neu. We compared neu-overexpressing Egr-1 knockout mice (neu/Egr-1 KO) to neu-overexpressing Egr-1 wild type or heterozygote mice (neu/Egr-1 WT or neu/Egr-1 het) with regard to onset of tumor appearance and number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, we established neu/Egr-1 WT and KO tumor cell lines derived from breast tumors developed in each mouse. Egr-1 deletion delayed tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays an oncogenic role in HER2/neu-driven mammary tumorigenesis.

MiR-551b-5p Contributes to Pathogenesis of Vein Graft Failure via Upregulating Early Growth Response-1 Expression.

Background:Vein graft failure (VGF) is a serious complication of coronary artery bypass graft, although the mechanism remains unclear. The study aimed to investigate the effects of microRNAs (miRNAs) on the endothelial dysfunction involved in VGF.Methods:Human umbilical vein endothelial cells (HUVECs) were subjected to mechanical stretch stimulation to induce endothelial dysfunction. Genome-wide transcriptome profiling was performed using the Human miRNA OneArray® V4 (PhalanxBio Inc., San Diego, USA). The miRNA-messenger RNA (mRNA) network was investigated using gene ontology and Kyoto Encyclopedia of Genes and Genomes. The miR-551b-5p mimic and inhibitor were applied to regulate miR-551b-5p expression in the HUVECs. The 5-ethynyl-2’-deoxyuridine assay, polymerase chain reaction (PCR), and Western blotting (WB) were used to assess HUVECs proliferation, mRNA expression, and protein expression, respectively. The vein graft model was established in early growth response (Egr)-1 knockout (KO) mice and wide-type (WT) C57BL/6J mice for pathological and immunohistochemical analysis. Endothelial cells isolated from the veins of WT and Egr-1 KO mice were subjected to mechanical stretch stimulation; PCR and WB were conducted to confirm the regulatory effect of Egr-1 on Intercellular adhesion molecule (Icam-1). One-way analysis of variance and independent t-test were performed for data analysis.Results:Thirty-eight miRNAs were differentially expressed in HUVECs after mechanical stretch stimulation. The bioinformatics analysis revealed that Egr-1 might be involved in VGF and was a potential target gene of miR-551b-5p. The mechanical stretch stimulation increased miR-551b-5p expression by 2.93 ± 0.08 fold (t = 3.07, P < 0.05), compared with the normal HUVECs. Transfection with the miR-551b-5p mimic or inhibitor increased expression of miR-551b-5p by 793.1 ± 171.6 fold (t = 13.84, P < 0.001) or decreased by 26.3% ± 2.4% (t = 26.39, P < 0.05) in the HUVECs, respectively. HUVECs proliferation and EGR-1 mRNA expression were significantly suppressed by inhibiting miR-551b-5p expression (P < 0.05). The lumens of the vein grafts in the Egr-1 KO mice were wider than that in the WT mice. Icam-1 expression was suppressed significantly in the Egr-1 KO vein grafts (P < 0.05).Conclusions:Increased miR-551b-5p expression leads to endothelial dysfunction by upregulating Egr-1 expression. EGR-1 KO can improve the function of a grafted vein through suppressing Icam-1.

Long-term acetaminophen treatment induced liver fibrosis in mice and the involvement of Egr-1.

Acetaminophen (APAP)-induced acute liver injury has already been well studied. However, whether long-term administration of APAP will cause liver fibrosis is still not very clear. This study aims to investigate the liver fibrosis in mice induced by long-term APAP treatment and the involvement of early growth response 1 (Egr-1). C57BL/6 mice were orally given with APAP (200, 300mg/kg) for 2, 6 or 10 weeks, respectively. Liver hydroxyproline content, collagen deposition and inflammatory cells infiltration were increased in mice treated with APAP (200, 300mg/kg) for 6 or 10 weeks. Liver mRNA expression of collagen (COL)1a1, Col3a1, transforming growth factor-β (TGF-β) and serum contents of COL1, COL3, TGF-β were all increased in APAP-treated mice. Liver expression of α-smooth muscle actin (α-SMA) and phosphorylated ERK1/2 and Smad2/3 were all increased in APAP-treated mice. Furthermore, increased liver mRNA expression of Egr-1 and its subsequent nuclear translocation were found in APAP-treated mice. Egr-1 knock-out mice were further applied. APAP-induced liver fibrosis was found to be more serious in Egr-1 knock-out mice. N-acetyl-p-benzoquinoneimine (NAPQI), the APAP hepatotoxic metabolite, increased cellular mRNA expression of α-SMA, Col1a1, Col3a1, TGF-β, induced ERK1/2 and Smad2/3 phosphorylation and Egr-1 nuclear translocation in hepatic stellate LX2 cells. In conclusion, long-term administration of APAP induced liver fibrosis in mice, and Egr-1 was critically involved in this process. This study points out a warning and reference for patients with long-term APAP ingestion in clinic.

A Sex‐Specific Role for Egr1 in Mediating Norepinephrine‐Induced Contraction in Mesenteric Arteries

The immediate early transcription factor early growth response 1 (Egr1) is upregulated in smooth muscle cells of atherosclerotic plaques and in adipose tissue of obese animals and humans. In a heterologous endothelial cell expression system, Egr1 overexpression upregulated the α2δ accessory subunit of the L‐type voltage gated Ca2+ channel (Fu et al, Gene; 2003). Moreover, Ca2+ influx via voltage‐gated Ca2+ channels increased Egr1 expression, suggesting that Egr1 regulates ion channel flux and vascular tone. In light of these findings, we tested the hypothesis that mesenteric arteries (MA; 2nd order) from Egr1 knockout (KO) mice would exhibit reduced contractility compared to MA from their wild‐type (WT) litter mates. Isolated vessel contractility was assessed using wire myography (DMT 620M). A small rightward shift in cumulative concentration response curves to norepinephrine (NE, 10−9 – 10−5M) in MA from male KO mice compared to WT littermates (−log EC50 = 5.96±0.11 vs −5.68±0.03 M NE, respectively; p=0.08) was observed, while there was no difference in NE‐induced contraction in female KO and WT mice. Neither maximal NE‐induced constriction nor maximal acetylcholine‐induced relaxation in NE‐preconstricted MA was different between male KO and WT mice or female KO and WT mice. Current studies are leveraging RNAseq to assess global transcriptome profiles of aorta from male and female WT and KO mice, to determine which pathways are impacted by Egr1. Our preliminary arterial contractility data suggest that smooth muscle Egr1 plays a role in modulating vascular tone.Support or Funding InformationThis work was funded by USDA‐ARS Project 6026‐51000‐010‐05S.

Early Growth Response-1 Plays an Important Role in Ischemia-Reperfusion Injury in Lung Transplants by Regulating Polymorphonuclear Neutrophil Infiltration.

Early growth response-1 (Egr-1) has been shown to be a trigger-switch transcription factor that is involved in lung ischemia-reperfusion injury (IRI). Mouse lung transplants were performed in wild-type (WT) C57BL/6 and Egr1-knockout (KO) mice in the following donor → recipient combinations: WT → WT, KO → WT, WT → KO, and KO → KO to determine whether the presence of Egr-1 in the donor or recipient is the most critical factor for IRI. Pulmonary grafts were retrieved after 18 hours of ischemia after 4 hours of reperfusion. We analyzed graft function by analyzing arterial blood gas and histology in each combination and assessed the effects of Egr1 depletion on inflammatory cytokines that are regulated by Egr-1 as well on polymorphonuclear neutrophil (PMN) infiltration. Deletion of Egr1 improved pulmonary graft function in the following order of donor → recipient combinations: WT → WT < WT → KO < KO → WT < KO → KO. Polymerase chain reaction assays for Il1B, Il6, Mcp1, Mip2, Icam1, and Cox2 showed significantly lower expression levels in the KO → KO group than in the other groups. Immunohistochemistry demonstrated clear Egr-1 expression in the nuclei of pulmonary artery endothelial cells and PMN cytoplasm in the WT grafts. Flow cytometry analysis showed that Egr1 deletion reduced PMN infiltration and that the extent of reduction correlated with graft function. Both graft and recipient Egr-1 played a role in lung IRI, but the graft side contributed more to this phenomenon through regulation of PMN infiltration. Donor Egr-1 expression in pulmonary artery endothelial cells may play an important role in PMN infiltration, which results in IRI after lung transplantation.

Early growth response 1 (Egr-1) directly regulates GABAA receptor α2, α4, and θ subunits in the hippocampus.

The homeostatic regulation of neuronal activity in glutamatergic and GABAergic synapses is critical for neural circuit development and synaptic plasticity. The induced expression of the transcription factor early growth response 1 (Egr-1) in neurons is tightly associated with many forms of neuronal activity, but the underlying target genes in the brain remained to be elucidated. This study uses a quantitative real-time PCR approach, in combination with invivo chromatin immunoprecipitation, and reveals that GABAA receptor subunit, GABRA2 (α2), GABRA4 (α4), and GABRQ (θ) genes, are transcriptional targets of Egr-1. Transfection of a construct that over-expresses Egr-1 in neuroblastoma (Neuro2A) cells up-regulates the α2, α4, and θ subunits. Given that Egr-1 knockout mice display less GABRA2, GABRA4, and GRBRQ mRNA in the hippocampus, and that Egr-1 directly binds to their promoters and induces mRNA expression, the present findings support a role for Egr-1 as a major regulator for altered GABAA receptor composition in homeostatic plasticity, in a glutamatergic activity-dependent manner. The early growth response 1 (Egr-1) is an inducible transcription factor to mediate rapid gene expression by neuronal activity. However, its underlying molecular target genes and mechanisms are not fully understood. We suggest that GABAA receptor subunits, GABRA2 (α2), GABRA4 (α4), and GABRQ (θ) genes are transcriptional targets of Egr-1. Neuronal activity-dependent up-regulation of Egr-1 might lead to altered subtypes of GABAA receptors for the maintenance of homeostatic excitatory and inhibitory balance for the regulation of synaptic strength.

STAT3 and Importins Are Novel Mediators of Early Molecular and Cellular Responses in Experimental Duodenal Ulceration

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directly upregulates VEGF, Ref-1, p21, and anti-apoptotic genes such as Bcl-xL. In this study, we hypothesized that STAT3 signaling is activated and provides a critical protective role that is required for enterocyte survival during the early phases of cysteamine-induced duodenal ulcers. We studied the effect of inhibition of STAT3 activity on cysteamine-induced duodenal ulcers in rats and egr-1 knockout mice using STAT3/DNA binding assay, immunohistochemistry, immunoblot, and quantitative reverse transcriptase PCR analyses. We found that G-quartet oligodeoxynucleotides T40214, a specific inhibitor of STAT3/DNA binding, aggravated cysteamine-induced duodenal ulcers in rats 2.8-fold (p<0.05). In the pre-ulcerogenic stage, cysteamine induced STAT3 tyrosine phosphorylation, its translocation to nuclei, an increased expression and nuclear translocation of importin α and β in the rat duodenal mucosa. Cysteamine enhanced the binding of STAT3 to its DNA consensus sequences at 6, 12, and 24h after cysteamine by 1.5-, 1.8-, and 3.5-fold, respectively, and activated the expression of STAT3 target genes such as VEGF, Bcl-xL, Ref-1, and STAT3-induced feedback inhibitor, a suppressor of cytokine signaling 3. We also demonstrated that egr-1 knockout mice, which are more susceptible to cysteamine-induced duodenal ulcers, had lower levels of STAT3 expression, its phosphorylation, expression of importin α or β, and STAT3/DNA binding than wild-type mice in response to cysteamine. Thus, STAT3 represents an important new molecular mechanism in experimental duodenal ulceration.

Impaired extinction of learned contextual fear memory in early growth response 1 knockout mice.

Inductive expression of early growth response 1 (Egr-1) in neurons is associated with many forms of neuronal activity. However, only a few Egr-1 target genes are known in the brain. The results of this study demonstrate that Egr-1 knockout (KO) mice display impaired contextual extinction learning and normal fear acquisition relative to wild-type (WT) control animals. Genome-wide microarray experiments revealed 368 differentially expressed genes in the hippocampus of Egr-1 WT exposed to different phases of a fear conditioning paradigm compared to gene expression profiles in the hippocampus of KO mice. Some of genes, such as serotonin receptor 2C (Htr2c), neuropeptide B (Npb), neuronal PAS domain protein 4 (Npas4), NPY receptor Y1 (Npy1r), fatty acid binding protein 7 (Fabp7), and neuropeptide Y (Npy) are known to regulate processing of fearful memories, and promoter analyses demonstrated that several of these genes contained Egr-1 binding sites. This study provides a useful list of potential Egr-1 target genes which may be regulated during fear memory processing.

Early growth response‐1 in the pathogenesis of cysteamineinduced duodenal ulcer

We showed earlier that the immediate‐early gene &amp; its zinc finger transcription factor early growth response (Egr) product were enhanced, including the expression of target genes (e.g., bFGF, PDGF, VEGF) in early stage of cysteamine (C)‐induced DU. Our recent studies demonstrated that deletion of egr‐1 gene led to 4.4‐fold increase in C‐induced DU in egr‐1 knockout (KO) vs. wild‐type (WT) mice. mRNA expression of bFGF, VEGF &amp; PDGF was still increased but protein expression of bFGF, VEGF &amp; PDGF was diminished in egr‐1 KO mice. We hypothesized that specific miRNAs in egr‐1 KO mice might be activated &amp; inhibit protein synthesis of egr‐1 target genes. Using miRXplore Microarray study we found that 11 miRNAs (e.g., mir‐696, mir‐467E, mir‐152 &amp; mir‐301B) were elevated by 1.8‐ to 1.9‐fold in egr‐1 KO mice vs. WT mice. Mir‐1892 decreased by 1.7‐fold in egr‐1 KO vs. WT mice. These miRNAs partially share validated targets relating to growth factor responses, e.g., FGFR3 &amp; VEGF. We also demonstrated that DU was significantly reduced in rats treated with adenovirus encoding egr‐1 on days 7 &amp; 14 (p &lt; 0.05) respectively. The mRNA &amp; protein expressions of egr‐1, bFGF, VEGF &amp; PDGF markedly increased in rats after egr‐1 gene therapy. We conclude that Egr‐1 plays a preventive role in the pathogenesis of DU &amp; further studies are needed to identify miRNA expression in egr‐1 KO &amp; WT mice with DU.(This work was supported by the Veterans Health Administration Merit Review Grant to S. Szabo).

Early growth response protein 1 promotes restenosis by upregulating intercellular adhesion molecule-1 in vein graft.

Objectives. To verify the relationship between Egr-1 and vein graft restenosis and investigate the related mechanisms. Methods. Mouse vein graft models were established in Egr-1 knockout (KO) and wild-type (WT) mice. The vein grafts in the mice were taken for pathological examination and immunohistochemical analysis. The endothelial cells (ECs) were stimulated by using a computer-controlled cyclic stress unit. BrdU staining and PCR were used to detect ECs proliferation activity and Egr-1 and ICAM-1 mRNA expression, respectively. Western-blot analysis was also used to detect expression of Egr-1 and intercellular adhesion molecule-1 (ICAM-1) proteins. Results. The lumens of vein grafts in Egr-1 KO mice were wider than in WT mice. ECs proliferation after mechanical stretch stimulation was suppressed by Egr-1 knockout (P < 0.05). Both in vein grafts and ECs from WT mice after mechanical stretch stimulation, mRNA expression and protein of Egr-1 and ICAM-1 showed increases (P < 0.05). However, ICAM-1 expression was significantly suppressed in ECs from Egr-1 knockout mice (P < 0.05). Conclusions. Egr-1 may promote ECs proliferation and result in vein graft restenosis by upregulating the expression of ICAM-1. As a key factor of vein graft restenosis, it could be a target for the prevention of restenosis after CABG surgery.

Differential Control of STIM1 Expression and Function by EGR Family Members in T Cells

Antigen presentation to T cells results in their activation through T Cell Receptor (TCR) stimulation, resulting in an intricate signaling web involving multiple steps and contributors. Elevation in cytosolic Ca2+ concentration is critical for T cell activation and requires Store Operated Ca2+ entry (SOCe). A key mediator of SOCe is the type 1A transmembrane protein Stromal Interaction Molecule 1 (STIM1) that stimulates Orai-mediated Ca2+ entry upon sensing ER Ca2+ store depletion. We have recently shown that STIM1 expression is regulated by a member of the Early Growth Response (EGR) family of zinc finger transcription factors, EGR1. However, examination of T cells collected from EGR1 knockout mice revealed a paradoxical increase in basal level STIM1 expression coinciding with increased expression of EGR4, another EGR family member. We further show that control of STIM1 expression, possibly by multiple EGRs, allows for a compensatory increase of SOCe in EGR1 knockout T cells that is not observed in other cell types such as fibroblasts. This data suggests that STIM1 expression and subsequently T cell function are dynamically regulated through interplay of EGR family members. Given the critical contribution of these Ca2+ signals to both T cell expansion and cytokine production, we anticipate that these findings have significant implications on the T cell activation process itself. We further predict that our findings have important implications towards the general understanding of transcriptional control of Ca2+ signaling.