Early Growth Response Gene Egr-1 Research Articles

A Single Dose of LSD Does Not Alter Gene Expression of the Serotonin 2A Receptor Gene (HTR2A) or Early Growth Response Genes (EGR1-3) in Healthy Subjects.

Rationale: Renewed interest has been seen in the use of lysergic acid diethylamide (LSD) in psychiatric research and practice. The repeated use of LSD leads to tolerance that is believed to result from serotonin (5-HT) 5-HT2A receptor downregulation. In rats, daily LSD administration for 4 days decreased frontal cortex 5-HT2A receptor binding. Additionally, a single dose of LSD acutely increased expression of the early growth response genes EGR1 and EGR2 in rat and mouse brains through 5-HT2A receptor stimulation. No human data on the effects of LSD on gene expression has been reported. Therefore, we investigated the effects of single-dose LSD administration on the expression of the 5-HT2A receptor gene (HTR2A) and EGR1-3 genes.Methods: mRNA expression levels were analyzed in whole blood as a peripheral biomarker in 15 healthy subjects before and 1.5 and 24 h after the administration of LSD (100 μg) and placebo in a randomized, double-blind, placebo-controlled, cross-over study.Results: LSD did not alter the expression of the HTR2A or EGR1-3 genes 1.5 and 24 h after administration compared with placebo.Conclusion: No changes were observed in the gene expression of LSD’s primary target receptor gene or genes that are implicated in its downstream effects. Remaining unclear is whether chronic LSD administration alters gene expression in humans.

Abstract A18: Inflaming breast cancer development: Expression of aromatase is increased by TNFα via the early growth response genes

Abstract Introduction: Breast cancer remains the leading cause of cancer-related death in Western women. In postmenopausal cases, up to 70% of tumors are classed as estrogen receptor positive (ER+), dependent on estrogen for continued growth and proliferative advantage. Adjuvant antiestrogen therapies are considered the cornerstone approach to the treatment of such tumors, and as such research is ongoing to maximize the effectiveness of drug treatments. The major source of estrogens for growth of ER+ breast cancers is local conversion of androgen precursors by the enzyme P450 aromatase. Inflammatory factors such as tumor necrosis factor-α (TNFα) stimulate transcription of the CYP19A1 gene that encodes aromatase, via its adipose-specific promoter I.4 (PI.4). The pathways by which this is achieved are not fully understood. Increased concentrations of TNFα are frequently detected within the stromal and epithelial compartments of an ER+ breast tumor microenvironment, contributing to a more invasive tumor fed by the increases in local estrogen production which are, in part, being driven by this critical cytokine. This study aims to identify the mechanisms underlying TNFα-dependent aromatase induction in the context of estrogen-dependent breast cancer. Methods: A custom gene array was performed to assess mRNA changes in primary human breast adipose fibroblasts (BAFs) that had been treated for 1 hr with 5ng/ml TNFα. This data was validated by qRT-PCR. Luciferase reporter assays were performed in C0S7 cells and EMSA assays were carried out using γP-32 detection. Results: A custom array showed that mRNA expression of the early growth response gene Egr1 was increased in BAFs that had been treated for 1 hr with TNFα. The family of early growth response genes Egr1, Egr2, Egr3 and Egr4 were all found to be significantly upregulated in a time-dependent manner in response to TNFα treatment, as validated by qRT-PCR. This occurs via induction of the NFkB pathway, as Egr mRNA induction was reduced in a dose-dependent manner upon pathway inhibition with the IKK-inhibiting compound BAY-11-7082. Egr2, Egr3 and Egr4 were able to induce Pl.4-driven luciferase reporter activity, with Egr2 proving to be the most potent activator. Serial 5′ deletion analysis of the 1 kb PI.4 promoter region revealed that the Egr2-induced promoter activity was mediated by a 41 bp sequence at the 3′ end, a region that did not contain any putative Egr binding sites. EMSA confirmed that there was no binding of any of the four Egr transcription factors to the 41 bp region. Mutagenesis of the 41 bp fragment is being carried out to narrow down the specific region of interest, and novel transcription factor binding sites will be confirmed with further luciferase assays and EMSA. Conclusion: We have elucidated a novel pathway by which TNFα is able to induce aromatase expression and thus estrogen biosynthesis in the breast via induction of the NFkB pathway and early growth response genes. Understanding TNFα signalling and the transcription factors it activates to turn on PI.4 CYP19A1 expression in BAFs is vital to the understanding of breast cancer pathology. These insights may help in the development of diagnostic tools or novel therapeutics in order to tackle the growing instances of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A18.

A new mechanism of gastric epithelial injury induced by acid exposure: The role of Egr‐1 and ERK signaling pathways

The molecular mechanisms by which gastric acid causes epithelial injury in the stomach and initiates an inflammatory reaction are poorly understood. We aimed in the present study to investigate the role of the early growth response gene Egr-1 and ERK in gastric epithelial cells following acid exposure, and the signaling pathways involved. Western blotting was used to assess Egr-1 protein levels in AGS cells. A quantitative measurement of acid-induced Egr-1 and ERK translocation was performed using a high content analysis approach. Egr-1 functionality was assessed by transient transfection with Egr-1 antisense oligonucleotide. Exposure of AGS cells to acidic conditions induced Egr-1 protein expression in a pH- and time-dependent manner. Egr-1 expression was markedly increased as the pH was reduced from pH 7.4 to 6.4. High content analysis of Egr-1 activation showed acid-induced Egr-1 nuclear translocation; a maximum observed at 1-2 h followed by a decline to basal levels beyond 4 h. Acidic pH also activated ERK1/2 phosphorylation, whereas ERK1/2 inhibitors PD98059 and U0216 blocked both acid-induced Egr-1 and ERK translocation and expression. Moreover, acid exposure up-regulated VEGF expression, which was inhibited by the Egr-1 antisense oligonucleotide. Our results also demonstrate that exposure to acid induces Egr-1 via MEK-ERK1/2 pathway. These data suggest that Egr-1 activation might play a crucial role in gastric mucosal inflammation and associated epithelial injury.

SDF1α/CXCR4 Signaling, via ERKs and the Transcription Factor Egr1, Induces Expression of a 67-kDa Form of Glutamic Acid Decarboxylase in Embryonic Hippocampal Neurons

Stromal cell-derived factor alpha (SDF1alpha) and its cognate receptor CXCR4 play an important role in neuronal development in the hippocampus, but the genes directly regulated by SDF1alpha/CXCR4 signaling are unknown. To study the role of CXCR4 targeted genes in neuronal development, we used neuronal cultures established from embryonic day 18 rats. Hippocampal neurons express CXCR4 receptor proteins and are stimulated by SDF1alpha resulting in activation of extracellular signal-regulated kinase (ERK)1/2 and the transcription factor cAMP-response element-binding protein. SDF1alpha rapidly induces the expression of the early growth response gene Egr1, a transcription factor involved in activity-dependent neuronal responses, in a concentration-dependent manner. Gel-shift analysis showed that SDF1alpha enhances DNA binding activity to the Egr1-containing promoter for GAD67. Chromatin immunoprecipitation analysis using an Egr1 antibody indicated that SDF1alpha stimulation increases binding of Egr1 to a GAD67 promoter DNA sequence. SDF1alpha stimulation increases the expression of GAD67 at both the mRNA and protein levels, and increases the amount and neurite localization of gamma-aminobutyric acid (GABA) in neurons already expressing GABA. SDF1alpha-induced Egr1/GAD67 expression is mediated by the G protein-coupled CXCR4 receptor and activation of the ERK pathway. Reduction of Egr1 gene expression using small interfering RNA technology lowers the level of GAD67 transcripts and inhibits SDF1alpha-induced GABA production. Inhibition of CXCR4 activation in the developing mouse brain in utero greatly reduced Egr1 and GAD67 mRNA levels and GAD67 protein levels, suggesting a pivotal role for CXCR4 signaling in the development of GABAergic neurons in vivo. Our data suggest that SDF1alpha/CXCR4/G protein/ERK signaling induces the expression of the GAD67 system via Egr1 activation, a mechanism that may promote the maturation of GABAergic neurons during development.

Suppression of tumor cell invasion by cyclooxygenase inhibitors is mediated by thrombospondin-1 via the early growth response gene Egr-1

Cyclooxygenase (COX) inhibitors have antitumorigenic activity and increase the expression of the early growth response gene Egr-1, a tumor suppressor gene and transcription factor. In this study, we have investigated the gene regulatory and anti-invasive activity of two traditional nonsteroidal anti-inflammatory drugs (NSAID), sulindac sulfide and indomethacin. These compounds inhibited tumor cell invasion and induced Egr-1 expression in lung adenocarcinoma A549 cells. Overexpression of Egr-1 reduced cellular invasion in the Matrigel system, whereas suppression of Egr-1 by small interference RNA (siRNA) attenuated the inhibition of Matrigel invasion by these compounds, indicating that Egr-1 is responsible for the decrease in invasion reported following treatment with NSAIDs. Egr-1-overexpressing cells were analyzed for genes involved in invasion and metastasis. Thrombospondin-1 (TSP-1) an antiangiogenic and anti-invasion protein was up-regulated by Egr-1 overexpression, which was confirmed following treatment with sulindac sulfide. Furthermore, the induction of TSP-1 by sulindac sulfide was blocked by Egr-1 siRNA. When TSP-1 was sequestered by the addition of anti-TSP-1 antibody, the inhibition of invasion by sulindac sulfide was attenuated, indicating that TSP-1 is involved in the inhibition of invasion by NSAIDs. We used the Min mouse model to determine if sulindac sulfide would increase Egr-1 and TSP-1 in vivo, because this model is widely used to study the effects of NSAIDs on tumor formation. Treatment of Min mice with concentrations of sulindac sulfide that inhibit tumor formation increased the expression of Egr-1 and TSP-1 in colonic tissues and in the polyps of these mice. This is the first report suggesting that COX inhibitors suppress tumor cell invasion via TSP-1, which occurs downstream of Egr-1.

Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKCα-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis.

Early Growth Response Factor-1 Mediates Prostaglandin E2-dependent Transcriptional Suppression of Cytokine-induced Tumor Necrosis Factor-α Gene Expression in Human Macrophages and Rheumatoid Arthritis-affected Synovial Fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic proinflammatory cytokine that modulates a broad range of inflammatory and immunological processes. We have investigated the potential immunomodulatory properties of prostaglandin E2 (PGE2) by examining the molecular mechanism by which the eicosanoid suppresses T-cell-derived interleukin-17 (IL-17)-induced TNF-alpha mRNA expression and protein synthesis in human macrophages and rheumatoid arthritis-affected synovial fibroblasts. Initial studies confirmed that PGE2 induces egr-1 mRNA expression and protein synthesis by restricted SAPK2/p38 MAPK-dependent activating transcription factor-2 (ATF-2) dimer transactivation of the egr-1 promoter as judged by studies using wild-type (WT) and deletion mutant egr-1 promoter constructs, Northern and Western blotting, and standard and supershift electrophoretic mobility shift analyses. Using human leukemic monocytic THP-1 cells stably transfected with WT and dominant-negative mutant expression constructs of Egr-1, cotransfected or not with a WT pTNF-615SVOCAT construct, we observed that PGE2 inhibition of IL-17-stimulated TNF-alpha mRNA expression and promoter activity was dependent on Egr-1 expression, as mutants of Egr-1, alone or in combination, markedly abrogated any inhibitory effect of PGE2. Standard and supershift electrophoretic mobility shift analysis, signaling "decoy" overexpression studies, and pTNF-615SVOCAT promoter assays using WT and mutant promoter constructs revealed that IL-17-up-regulated promoter activity was largely dependent on ATF-2/c-Jun transactivation. PGE2 suppression of IL-17-induced ATF-2/c-Jun transactivation and DNA binding was dependent on Egr-1-mediated inhibition of induced c-Jun expression. We suggest that egr-1 is an immediate-early PGE2 target gene that may be a key regulatory factor in mediating eicosanoid control of genes involved in the immune and inflammatory responses.

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1 h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.

Expression of NAG-1, a Transforming Growth Factor-β Superfamily Member, by Troglitazone Requires the Early Growth Response Gene EGR-1

Troglitazone (TGZ) and 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2) are peroxisome proliferator-activated receptor-gamma (PPARgamma) ligands that have been shown to possess pro-apoptotic activity in human colon cancer. Although these compounds bind to PPARgamma transcription factors as agonists, emerging evidence suggests that TGZ acts independently of PPARgamma in many functions, including apoptosis. We previously reported that TGZ induces an early growth response transcription factor (EGR-1) by the ERK phosphorylation pathway rather than by the PPARgamma pathway (Baek, S. J., Wilson, L. C., Hsi, L. C., and Eling, T. E. (2003) J. Biol. Chem. 278, 5845-5853). In this report, we show that the expression of the antitumorigenic and/or pro-apoptotic gene NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) is induced by TGZ and correlates with EGR-1 induction. In cotransfection and gel shift assays, we show that EGR-1-binding sites are located within region -73 to -51 of the NAG-1 promoter and have an important role in the transactivation of TGZ-induced NAG-1 expression. In contrast, PGJ2 induced NAG-1 protein expression, but PGJ2 may not affect the same region that TGZ does in the NAG-1 promoter. The effect of PGJ2 is probably PPARgamma-dependent because a PPARgamma antagonist inhibited the PGJ2-induced expression of NAG-1. TGZ-induced NAG-1 expression was not inhibited by the PPARgamma antagonist. The fact that TGZ-induced NAG-1 expression was accompanied by the biosynthesis of EGR-1 also suggests that EGR-1 plays a pivotal role in TGZ-induced NAG-1 expression. Our results suggest that EGR-1 induction is a unique property of TGZ, but is independent of PPARgamma activation. The up-regulation of NAG-1 may provide a novel explanation for the antitumorigenic property of TGZ.

Induction of Egr-1 mRNA and protein by endothelin 1, angiotensin II and norepinephrine in neonatal cardiac myocytes.

The early growth response gene Egr-1 is a nuclear transcription factor known to serve as an intermediary in a broad range of signal transduction processes. Recent studies have assigned Egr-1 a new role as an amplifier of gene expression. Egr-1 mRNA is expressed in the myocardium and is rapidly induced in response to hypertrophic stimuli. However, induction of the Egr-1 protein has not yet been demonstrated in the myocardium; on the other hand, in skeletal muscle cells we have shown translational regulation of Egr-1. To further investigate the role of Egr-1 in the regulatory mechanisms of a variety of signal transduction processes we have therefore asked whether bona fide hypertrophic stimuli induce Egr-1 protein subsequently to its mRNA in neonatal rat cardiomyocytes or whether translational block occurs. In confocal laser studies the Egr-1 protein was nuclearly localized. Norepinephrine (NE, 2 microM), angiotensin II (AII, 0.1 microM), and endothelin 1 (E1, 0.1 microM) each induced the Egr-1 mRNA 6-8 fold and the Egr-1 protein 3-5 fold (n = 3, p < 0.01). Therefore, in contrast to skeletal muscle cells, these stimuli increased Egr-1 mRNA and protein levels. These results point further to the role of Egr-1 as a possible amplifier of signal transduction in the myocardium.

Adenine dinucleotides: a novel class of signalling molecules.

1. Adenine dinucleotides (Ap3A, Ap4A, Ap5A, Ap6A) are stored in secretory granules of thrombocytes, chromaffin cells and neuronal cells. After release into the extracellular space, the dinucleotides exhibit divergent biological effects on a variety of target cells and organs. The dinucleotides are metabolized by soluble enzymes in the blood plasma as well as by membrane-bound ectoenzymes of endothelial cells, smooth muscle cells, and other cell types. 2. The enzymatic cleavage of the dinucleotides plays a dual role for their biological function: (a) termination of the signal; and (b) generation of purinergically active products such as ATP, ADP and finally adenosine. In contrast to ATP the dinucleotides are long-lived purine nucleotides in the blood. 3. The potential role of the dinucleotides as signalling molecules has been demonstrated in several systems. The adenosine polyphosphates have autocrine function for thrombocytes. Ap3A at low concentration reversibly activates isolated platelets. The mechanism of activation has been elucidated by showing a continuous cleavage of Ap3A, leading to the formation of ADP which is a known agonist of the P2T receptor on thrombocytes. Ap4A and other dinucleotides act as antagonists and inhibit platelet activation. 4. The vasotone of perfused isolated arteries as well as of resistance vessels in the beating heart is differentially influenced by adenine dinucleotides. While Ap3A and Ap4A exhibit relaxing effects at micromolar concentrations, Ap5A and Ap6A elicit vasoconstriction in these vessels. 5. In rat kidney mesangial cells adenine dinucleotides efficiently promote growth. Stimulation of DNA synthesis by various growth factors is enhanced synergistically. ApnA significantly increase the expression of the early growth response gene Egr-1. 6. The specificity and, in some tissues, the uniqueness of effects evoked by dinucleotides may be mediated by genuine dinucleotide receptors (P4) or by specialized P2 receptors (P2D).

Transmembrane signaling by an insulin receptor lacking a cytoplasmic beta-subunit domain.

To assess the function of the cytoplasmic domain of the insulin receptor (IR) beta subunit, we have studied a mutant IR truncated by 365 aa (HIR delta 978), thereby deleting > 90% of the cytoplasmic domain. HIR delta 978 receptors were processed normally to homodimers that were expressed at the cell surface where they bind insulin with normal affinity. Although these truncated IRs were inactive with respect to ligand-induced internalization and autophosphorylation, insulin stimulated endogenous substrate (pp185) phosphorylation significantly more in HIR delta 978 cells than in untransfected Rat1 cells. Importantly, despite absence of the beta-subunit cytoplasmic domain, fibroblasts expressing HIR delta 978 receptors displayed enhanced sensitivity to insulin for stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, thymidine incorporation, and S6 kinase activity compared with parental fibroblasts. Insulin also induced the expression of the protooncogene c-fos and the early growth response gene Egr-1 in HIR delta 978 cells far greater than in parental Rat1 fibroblasts. Furthermore, an agonistic monoclonal antibody specific for the human IR stimulated insulin action in fibroblasts expressing wild-type human IR but had no effect on HIR delta 978 cells. In conclusion, the HIR delta 978 truncated IRs appear to confer enhanced insulin sensitivity by augmenting the signaling properties of the endogenous rodent IRs.

Effect of angiotensin II and norepinephrine on early growth response genes in the rat kidney

Angiotensin II (Ang II) has growth promoting effects in a number of cells and tissues in vitro. The purpose of this study was to examine the in vivo effect of Ang II on the renal expression of the early growth response genes c-fos, Egr-1 and c-jun. Adult male rats underwent two basal 30 minute clearance periods during which the normal saline vehicle was infused into the left renal artery. Normal saline, Ang II (50 ng/kg/min), or Ang II plus the Ang II antagonist Sar1 Gly8-angiotensin II (10 micrograms/kg/min) were then selectively infused into the left renal artery for two additional 30 minute periods. MAP was similar during basal and Ang II infusion. Hemodynamic effects (decrease in GFR and RPF and increase in renal vascular resistance) were observed only in the Ang II-infused left kidney allowing the right kidney to serve as a paired control for the effects of anesthesia and surgery. Significant increases in the expression of the early growth response genes Egr-1 and c-fos, but not c-jun, were found in the Ang II-infused left kidney compared to the control right kidney. The simultaneous infusion of Ang II (50 ng/kg/min) and the Ang II antagonist Sar1 Gly8-angiotensin II (10 micrograms/kg/min) blocked the increase in Egr-1 and c-fos expression, demonstrating the specificity of the response to Ang II. To compare the effect of another renal vasoconstrictor on the expression of early growth response genes, norepinephrine (40 ng/kg/min) was infused into the left renal artery.(ABSTRACT TRUNCATED AT 250 WORDS)

CDNA sequence of the human cellular early growth response gene Egr-1

cDNA sequence of the human cellular early growth response gene Egr-1