Biosynthesis Of Egr-1 Research Articles

Early growth response-1: Key mediators of cell death and novel targets for cardiovascular disease therapy.

Cardiovascular diseases are seen to be a primary cause of death, and their prevalence has significantly increased across the globe in the past few years. Several studies have shown that cell death is closely linked to the pathogenesis of cardiovascular diseases. Furthermore, many molecular and cellular mechanisms are involved in the pathogenesis of the cardiac cell death mechanism. One of the factors that played a vital role in the pathogenesis of cardiac cell death mechanisms included the early growth response-1 (Egr-1) factor. Studies have shown that abnormal Egr-1 expression is linked to different animal and human disorders like heart failure and myocardial infarction. The biosynthesis of Egr-1 regulates its activity. Egr-1 can be triggered by many factors such as serum, cytokines, hormones, growth factors, endotoxins, mechanical injury, hypoxia, and shear stress. It also displays a pro-apoptotic effect on cardiac cells, under varying stress conditions. EGR1 mediates a broad range of biological responses to oxidative stress and cell death by combining the acute changes occurring in the cellular environment with sustained changes in gene expression. The primary regulatory role played by the Egr-1-targeting DNAzymes, microRNAs, and oligonucleotide decoy strategies in cardiovascular diseases were identified to provide a reference to identify novel therapeutic targets for cardiovascular diseases.

Glucose Homeostasis and Pancreatic Islet Size Are Regulated by the Transcription Factors Elk-1 and Egr-1 and the Protein Phosphatase Calcineurin.

Pancreatic β-cells synthesize and secrete insulin. A key feature of diabetes mellitus is the loss of these cells. A decrease in the number of β-cells results in decreased biosynthesis of insulin. Increasing the number of β-cells should restore adequate insulin biosynthesis leading to adequate insulin secretion. Therefore, identifying proteins that regulate the number of β-cells is a high priority in diabetes research. In this review article, we summerize the results of three sophisticated transgenic mouse models showing that the transcription factors Elk-1 and Egr-1 and the Ca2+/calmodulin-regulated protein phosphatase calcineurin control the formation of sufficiently large pancreatic islets. Impairment of the biological activity of Egr-1 and Elk-1 in pancreatic β-cells leads to glucose intolerance and dysregulation of glucose homeostasis, the process that maintains glucose concentration in the blood within a narrow range. Transgenic mice expressing an activated calcineurin mutant also had smaller islets and showed hyperglycemia. Calcineurin induces dephosphorylation of Elk-1 which subsequently impairs Egr-1 biosynthesis and the biological functions of Elk-1 and Egr-1 to regulate islet size and glucose homeostasis.

Expression of the Transcription Factor Egr-1 in Pancreatic Acinar Cells Following Stimulation of Cholecystokinin or Gαq-Coupled Designer Receptors

Backgound/Aims: The injection of cerulein, an analogue of the pancreatic secretagogue cholecystokinin (CCK), induces acute pancreatitis in mice that is accompanied by the synthesis of the transcription factor Egr-1. The signaling cascade that connects cerulein stimulation with enhanced Egr-1 biosynthesis was analyzed. Methods: AR42J rat pancreatic acinar cells were used as a model system to measure cerulein-induced Egr-1 biosynthesis. For comparison, the signaling cascade induced by activation of Gαq-coupled designer receptors with the designer drug clozapine-N-oxide (CNO) was investigated. Results: Stimulation of AR42J cells with cerulein induced a robust and transient biosynthesis of Egr-1. The signaling cascade connecting cerulein stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca²⁺ and the activation of the protein kinases PKC, Raf and ERK, while expression of MKP-1 prevented Egr-1 biosynthesis in cerulein-stimulated AR42J cells. In addition, ternary complex factors are required to connect cerulein stimulation with enhanced transcription of the Egr-1 gene. Egr-1 biosynthesis induced in CNO-stimulated AR42J pancreatic acinar cells expressing Gαq-coupled designer receptors required identical signaling molecules, although subtle differences were observed in comparison to cerulein/CCK receptor signaling. Conclusion: We propose that overstimulation of the canonical Gαq-induced signaling pathway may be crucial for inducing acute pancreatitis.

Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells: ACTIVATION OF EGR-1 EXPRESSION INVOLVING TRPM3, VOLTAGE-GATED CALCIUM CHANNELS, ERK, AND TERNARY COMPLEX FACTORS

The neurosteroid pregnenolone sulfate acts on the nervous system by modifying neurotransmission and receptor functions, thus influencing synaptic strength, neuronal survival, and neurogenesis. Here we show that pregnenolone sulfate induces a signaling cascade in insulinoma cells leading to enhanced expression of the zinc finger transcription factor Egr-1 and Egr-1-responsive target genes. Pharmacological and genetic experiments revealed that influx of Ca(2+) ions via transient receptor potential M3 and voltage-gated Ca(2+) channels, elevation of the cytosolic Ca(2+) level, and activation of ERK are essential for connecting pregnenolone sulfate stimulation with enhanced Egr-1 biosynthesis. Expression of a dominant-negative mutant of Elk-1, a key regulator of gene transcription driven by a serum response element, attenuated Egr-1 expression following stimulation, indicating that Elk-1 or related ternary complex factors connect the transcription of the Egr-1 gene with the pregnenolone sulfate-induced intracellular signaling cascade elicited by the initial influx of Ca(2+). The newly synthesized Egr-1 was biologically active and bound under physiological conditions to the regulatory regions of the Pdx-1, Synapsin I, and Chromogranin B genes. Pdx-1 is a major regulator of insulin gene transcription. Accordingly, elevated insulin promoter activity and increased mRNA levels of insulin could be detected in pregnenolone sulfate-stimulated insulinoma cells. Likewise, the biosynthesis of synapsin I, a synaptic vesicle protein that is found at secretory granules in insulinoma cells, was stimulated in pregnenolone sulfate-treated INS-1 cells. Together, these data show that pregnenolone sulfate induces a signaling cascade in insulinoma cells that is very similar to the signaling cascade induced by glucose in β-cells.

Epidermal-growth-factor-induced proliferation of astrocytes requires Egr transcription factors

Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

BackgroundThe serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts.ResultsStimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK). Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M1 muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not.ConclusionThis study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca2+-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.

Calcium influx into MIN6 insulinoma cells induces expression of Egr-1 involving extracellular signal-regulated protein kinase and the transcription factors Elk-1 and CREB

Glucose induces many changes in the transcriptional pattern of β-cells derived from the endocrine pancreas. The zinc finger protein Egr-1 belongs to the transcription factors that are activated in glucose-treated β-cells. Egr-1 expression is additionally induced by treatment of MIN6 pancreatic β-cells with tolbutamide, a compound that triggers a closure of ATP-dependent potassium channels, K ATP, in the plasma membrane or by KCl that depolarizes the cell membrane. Stimulation with glucose, tolbutamide or KCl induces a Ca 2+ influx into the β-cells via L-type Ca 2+ channels. Accordingly, incubation of the cells with the L-type Ca 2+ channel blocker nifedipine or the acetoxymethylester of the cytosolic Ca 2+ chelator BAPTA prevented Egr-1 expression. Moreover, diacylgycerol-dependent protein kinase C isoenzymes and activation of extracellular signal-regulated protein kinase (ERK) are required for glucose-, tolbutamide- and KCl-induced Egr-1 expression. The signaling cascade was blocked by MAP kinase phosphatase-1 (MKP-1) overexpression that dephosphorylated ERK in the nucleus. Stimulation of β-cells by glucose, tolbutamide and KCl induced the phosphorylation of the transcription factors Elk-1 and CREB. ChIP experiments revealed that phosphorylated Elk-1 and CREB bound under physiological conditions to the Egr-1 gene. Lentiviral-mediated expression of dominant-negative mutants of Elk-1 or CREB interfered with glucose-, tolbutamide- and KCl-induced upregulation of Egr-1 biosynthesis. Together, these data indicate that stimulus-induced transcription of the Egr-1 gene in β-cells requires combinatorial regulation by Elk-1 and CREB following activation of ERK. The newly synthesized Egr-1 is biologically active and binds under physiological conditions to the genes encoding basic fibroblast growth factor, tumor necrosis factor α, transforming growth factor β and PTEN.

Transcriptional response to muscarinic acetylcholine receptor stimulation: Regulation of Egr-1 biosynthesis by ERK, Elk-1, MKP-1, and calcineurin in carbachol-stimulated human neuroblastoma cells

Carbachol-mediated activation of type M 3 muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5′-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M 3 muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.

Transcriptional response to muscarinic acetylcholine receptor stimulation: Regulation of Egr-1 biosynthesis by Elk-1, MKP-1 and calcineurin in carbachol-stimulated human neuroblastoma cells

Transcriptional response to muscarinic acetylcholine receptor stimulation: Regulation of Egr-1 biosynthesis by Elk-1, MKP-1 and calcineurin in carbachol-stimulated human neuroblastoma cells

P2X7 receptor stimulation upregulates Egr‐1 biosynthesis involving a cytosolic Ca2+ rise, transactivation of the EGF receptor and phosphorylation of ERK and Elk‐1

The P2X(7) receptor is an ATP-gated ionotropic receptor that is permeable for small cations including Ca(2+) ions. Using 293 cells expressing P2X(7) receptors, we show that the P2X(7) receptor-specific ligand 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) induces a signaling cascade leading to the biosynthesis of biologically active Egr-1, a zinc finger transcription factor. BzATP-triggered Egr-1 biosynthesis was attenuated by the mitogen-activated protein kinase kinase inhibitor PD98059, by BAPTA-AM, the acetoxymethylester of the cytosolic Ca(2+) chelator BAPTA, and by an epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor (AG1478). These results indicate that phosphorylation and activation of extracellular signal-regulated protein kinase ERK, elevated levels of intracellular Ca(2+) and the transactivation of the EGF receptor are essential for BzATP-induced upregulation of Egr-1. The requirement of Ca(2+) within the signaling cascade was upstream of Raf kinase activation. Lentiviral-mediated expression of MAP kinase phosphatase-1 (MKP-1), a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, inhibited Egr-1 biosynthesis following BzATP stimulation, indicating that MKP-1 functions as a nuclear shut-off device. Furthermore, the ternary complex factor Elk-1 was phosphorylated and the transcriptional activation potential of Elk-1 was enhanced following P2X(7) receptor stimulation. Expression of a dominant-negative mutant of Elk-1 impaired BzATP-induced upregulation of Egr-1 biosynthesis. Thus, Elk-1 connects the intracellular signaling cascade elicited by activation of P2X(7) receptors with the transcription of the Egr-1 gene.

Upregulation of Egr-1 biosynthesis in pituitary gonadotropes following activation of GnRH and muscarinic acetylcholine receptors

Stimulation of pituitary gonadotropes expressing the Gq/ 11-coupled gonadotropin releasing hormone (GnRH) and muscarinic M3 acetylcholine receptors with GnRH or carbachol upregulates the biosynthesis of the zinc finger transcription factor Egr-1. Receptor stimulation activates phospholipase C, leading to elevated cytosolic Ca2+ levels following stimulation. Accordingly, GnRHand carbachol-triggered biosynthesis of Egr-1 was blocked by BAPTA-AM, an intracellular calcium chelator. Ca2+-mediated activation of extracellular signal-regulated protein kinase (ERK) was accomplished via activation of protein kinase C and transactivation of the EGF receptor. Calmodulin was also required to connect GnRH and carbachol signaling with the upregulation of Egr-1 gene transcription. Lentiviral-mediated expression of a dominant-negative mutant of Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, impaired GnRHand carbachol-induced biosynthesis of Egr-1. Thus, Elk-1 connects the GnRHand carbacholinduced signaling cascades in the nucleus with the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1 (MKP-1), the enzyme that dephosphorylates and inactivates ERK, completely blocked Egr-1 biosynthesis following GnRH or carbachol stimulation, indicating that MKP-1 functions as a nuclear shut-of-device of these signaling pathways. Chromatin immunoprecipitation experiments revealed that Egr-1 bound in vivo to the regulatory regions of the basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGFbeta) genes following stimulation of the cells with GnRH or carbachol. As a result, elevated levels of bFGF and TGFbeta mRNA were detected in GnRH and carbachol stimulated gonadotropes, corroborating the view that Egr-1 transactivates the bFGF and TGFbeta genes following stimulation of the cells with GnRH or carbachol. from Annual Meeting of the Study Group Neurochemistry. International Conference of the Gesellschaft fur Biochemie und Molekularbiologie 2006 (GBM 2006): Molecular pathways in health and disease of the nervous system Witten, Germany. 28–30 September 2006

Upregulation of Egr-1 biosynthesis in human neuroblastoma cells following activation of epidermal growth factor or muscarinic acetylcholine receptors

The zinc finger transcription factor Egr-1 controls many biological functions in the nervous system, including neuronal plasticity, cell death and proliferation. Egr-1 connects cellular signaling cascades with changes of the gene expression pattern. Here, we show that epidermal growth factor (EGF) and carbachol stimulated the biosynthesis of Egr-1 in human neuroblastoma cells. The analysis of a lentiviral transmitted Egr-1-responsive reporter gene, embedded into the chromatin, revealed that Egr-1-dependent transcription was upregulated following stimulation. The signalling cascade initiated by carbachol involved the muscarinic acetylcholine receptors type I and III and the activation of diacylgycerol-dependent protein kinase C isoforms. Both signaling cascades, initiated by EGF or carbachol, required the activation of extracellular signal-regulated protein kinase ERK. In addition, we observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Elk 1, a key transcriptional regulator of serum response element-driven gene transcription. Incubation of neuroblastoma cells with EGF or carbachol also triggered an upregulation of MAP kinase phosphatase-1 (MKP-1) that dephosphorylates and inactivates ERK in the nucleus. Lentiviral-mediated expression of MKP-1 completely blocked Egr-1 biosynthesis following EGF or carbachol stimulation, indicating that MKP-1 functions as a nuclear shut-ofdevice of EGF or carbachol-induced Egr-1 gene transcription in neuroblastoma cells. from Annual Meeting of the Study Group Neurochemistry. International Conference of the Gesellschaft fur Biochemie und Molekularbiologie 2006 (GBM 2006): Molecular pathways in health and disease of the nervous system Witten, Germany. 28–30 September 2006

Transcriptional activation of the Egr-1 gene mediated by tetradecanoylphorbol acetate and extracellular signal-regulated protein kinase

Activation of extracellular signal-regulated protein kinase (ERK) triggers the biosynthesis of Egr-1, a zinc finger transcription factor. Likewise, the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) strongly upregulates Egr-1 biosynthesis. Here, we have analyzed the genetic elements involved in the regulation of Egr-1 gene transcription by ERK and TPA in human hepatoma cells. Expression experiments using mitogen-activated protein kinase phosphatase-1 or a dominant-negative mutant of the ternary complex factor Elk-1 revealed that the distal cluster of serum response elements is essential in the TPA-induced enhancement of Egr-1 promoter activity, encompassing two independent TPA-responsive elements. The CRE in the proximal Egr-1 promoter plays, if anything, only a marginal role in TPA-induced stimulus–transcription coupling of the Egr-1 gene. The fact that Egr-1 promoter/reporter gene transcription is upregulated by a constitutively active CREB mutant indicates that the CRE couples other signaling cascades via CREB to the Egr-1 gene.

The zinc finger transcription factor Egr-1 is upregulated in arsenite-treated human keratinocytes.

Arsenite is a human carcinogen that may induce cancer in skin, liver, kidney, bladder or lung. Arsenite executes its toxic effects by the induction of signaling cascades. In particular, the activation of the stress-induced protein kinase c-Jun N-terminal protein kinase and p38 and the phosphorylation and activation of the transcription factor c-Jun have been linked to the biological effects of arsenite. We analyzed whether arsenite has an impact on the biosynthesis of the zinc finger transcription factor Egr-1. Egr-1 transcription is upregulated following treatment of cells with hormones, cytokines or toxic chemicals, and thus Egr-1 integrates many signaling cascades with changes in gene expression patterns. Here, we show by Western blot experiments that arsenite induces a transient synthesis of Egr-1 in human HaCaT keratinocytes. Egr-1 biosynthesis was activated by arsenite concentrations insufficient for the induction of c-Jun biosynthesis. This arsenite-triggered Egr-1 biosynthesis was completely inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 and by AG1487, an epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor. These results indicate that activation of the EGF receptor as well as stimulation of the mitogen activated/extracellular signal-regulated protein kinase is essential for arsenite-induced upregulation of Egr-1. Moreover, we detected an elevated transcriptional activation potential of the ternary complex factor Elk1, a key transcriptional regulator of serum response element-driven gene transcription. The Egr-1 5'-flanking region contains five serum response elements. Accordingly, we observed an increase in Egr-1 promoter activity as a result of arsenite treatment. The fact that low concentrations of arsenite are sufficient to induce Egr-1 biosynthesis suggests that Egr-1 may be an integral part of arsenite-triggered signaling cascades leading to tumor formation or cell death via alterations of the cellular genetic program.

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.

Troglitazone, a Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Selectively Induces the Early Growth Response-1 Gene Independently of PPARγ: A NOVEL MECHANISM FOR ITS ANTI-TUMORIGENIC ACTIVITY

Troglitazone (TGZ) is a peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand that has pro-apoptotic activity in human colon cancer. Although TGZ binds to PPAR gamma transcription factors as an agonist, emerging evidence suggests that TGZ acts independently of PPAR gamma in many functions, including apoptosis. Early growth response-1 (Egr-1) transcription factor has been linked to apoptosis and shown to be activated by extracellular signal-regulated kinase (ERK). We investigated whether TGZ-induced apoptosis may be related to Egr-1 induction, because TGZ has been known to induce ERK activity. Our results show that Egr-1 is induced dramatically by TGZ but not by other PPAR gamma ligands. TGZ affects Egr-1 induction at least by two mechanisms; TGZ increases Egr-1 promoter activity by 2-fold and prolongs Egr-1 mRNA stability by 3-fold. Inhibition of ERK phosphorylation in HCT-116 cells abolishes the Egr-1 induction by TGZ, suggesting its ERK-dependent manner. Further, the TGZ-induced Egr-1 expression results in increased promoter activity using a reporter system containing four copies of Egr-1 binding sites, and TGZ induces Egr-1 binding activity to Egr-1 consensus sites as assessed by gel shift assay. In addition, TGZ induces ERK-dependent phosphorylation of PPAR gamma, resulting in the down-regulation of PPAR gamma activity. The fact that TGZ-induced apoptosis is accompanied by the biosynthesis of Egr-1 suggests that Egr-1 plays a pivotal role in TGZ-induced apoptosis in HCT-116 cells. Our results suggest that Egr-1 induction is a unique property of TGZ compared with other PPAR gamma ligands and is independent of PPAR gamma activation. Thus, the up-regulation of Egr-1 may provide an explanation for the anti-tumorigenic properties of TGZ.

Substance P induced biosynthesis of the zinc finger transcription factor Egr-1 in human glioma cells requires activation of the epidermal growth factor receptor and of extracellular signal-regulated protein kinase

Substance P is a member of the tachykinin family of neuropeptides that plays an important role in pain transmission, neurogenic inflammatory diseases and the adaptive response to stress. Substance P exerts its biological activities via binding to a G-protein coupled receptor of the neurokinin (NK) receptor family. Here, we show by Western blot experiments that substance P induced a transient synthesis of the zinc finger transcriptional regulator Egr-1 in human glioma cells. Substance P-induced stimulation of Egr-1 biosynthesis was completely inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 and by AG1487, an epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor. These results indicate that transactivation of the EGF receptor as well as stimulation of the mitogen activated/extracellular signal-regulated protein kinase (ERK) are essential for substance P/NK-1 receptor-induced activation of Egr-1 biosynthesis. Moreover, we show that the signaling cascade initiated by substance P or EGF are indistinguishable, including the activation of the EGF receptor, the activation of ERK, and the final stimulation of Egr-1 biosynthesis. The synthesis of Egr-1 in glioma cells as a result of substance P stimulation suggests that substance P exerts long-term effects in glioma cells via Egr-1-mediated gene transcription.

Neuronal cell death induced by antidepressants: lack of correlation with Egr-1, NF-κB and extracellular signal-regulated protein kinase activation

The tricyclic antidepressants (TCA) amitriptyline and desipramine and the serotonin reuptake inhibitor fluoxetine induce, at μM concentrations, cell death in HT22 immortalized hippocampal neurons and PC12 pheochromocytoma cells. Here, we show that these neurotoxic effects are accompanied by a selective activation of extracellular signal-regulated protein kinase (ERK), the biosynthesis of the transcription factor Egr-1 and an increase in the transcriptional activity of NF-κB. However, an impairment of both ERK activation and Egr-1 biosynthesis by the MAP kinase kinase-1 (MEK-1) inhibitor PD98059 did not block cell death. Moreover, stimulation of ERK phosphorylation and Egr-1 biosynthesis by sphingosine-1-phosphate did not induce cell death, indicating that stimulation of the ERK signaling pathway and Egr-1 biosynthesis are not required for neuronal cell death induced by antidepressants. Likewise, attenuation of antidepressant-induced NF-κB activity by elevation of the intracellular cAMP concentration or by retroviral driven expression of the non-degradable superrepressor IκBαS32A/S36A demonstrated that the elevation of NF-κB activity by amitriptyline, desipramine and fluoxetine is not an integral part of the apoptotic signaling cascade triggered by these compounds.

Nitric oxide-induced programmed cell death in human neuroblastoma cells is accompanied by the synthesis of Egr-1, a zinc finger transcription factor.

Nitric oxide (NO) is cytotoxic for human SH-SY5Y neuroblastoma cells. While nuclear condensation was visible in cells treated with nitric oxide donors, we observed that the plasma membrane remained intact, indicating that NO induced apoptotic cell death. We analyzed the NO-induced apoptotic signaling cascade in SH-SY5Y cells and observed a striking increase in early growth response (Egr)-1 promoter activity as a result of NO-induced cell death. Likewise, we detected an activation of the transcriptional activation potential of the ternary complex factor Elk1, a key transcriptional regulator of serum response element-driven gene transcription. Egr-1 is a zinc finger transcription factor that couples extracellular signals to long-term responses by altering expression of Egr-1 target genes. The Egr-1 5'-flanking region contains five serum response elements (SRE) that function as genetic elements for stimulus-transcription coupling. Moreover, these SREs are binding sites for Elk1, suggesting that NO activated Egr-1 gene transcription via activation of Elk1. The NO-induced biosynthesis of Egr-1 was confirmed by Western blot analysis and an NO-dependent increase in the transcriptional activation potential of Egr-1 was observed. The fact that NO-induced neuronal cell death is accompanied by the biosynthesis of Egr-1 suggests that Egr-1 may be an integral part of the NO triggered apoptotic signaling cascade.

Epidermal growth factor and thrombin induced proliferation of immortalized human keratinocytes is coupled to the synthesis of Egr-1, a zinc finger transcriptional regulator.

The epidermal growth factor (EGF) receptor is highly expressed in HaCaT keratinocytes as shown by Western blotting. Stimulation of HaCaT cells with EGF, and also with the serine protease thrombin, induced DNA synthesis, measured by incorporation of 5-bromo-2'-deoxyuridine into the DNA of proliferating cells. Using antibodies directed against the active form of the EGF receptor, we show that in HaCaT cells EGF and thrombin triggered a rapid activation of the EGF receptor, followed by the phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK). Moreover, EGF and thrombin induced a transient synthesis of the zinc finger transcriptional regulator Egr-1. Proliferation, activation of ERK, and biosynthesis of Egr-1 was completely inhibited in EGF or thrombin-treated HaCaT cells by the MAP kinase kinase inhibitor PD98059 and by AG1487, an EGF receptor-specific tyrosine kinase inhibitor. These data indicate that phosphorylation and activation of both the EGF receptor and ERK are essential for mitogenic signaling via EGF and thrombin. The synthesis of Egr-1 in HaCaT cells as a result of EGF or thrombin stimulation suggests that Egr-1 may be an important "late" part of the EGF and thrombin-initiated signaling cascades. We postulate that Egr-1 may function as a "third messenger" in keratinocytes connecting mitogenic stimulation with changes in gene transcription.