Egr-1 Protein Levels Research Articles

Up-regulation of Egr1 by 1,25-dihydroxyvitamin D3 contributes to increased expression of p35 activator of cyclin-dependent kinase 5 and consequent onset of the terminal phase of HL60 cell differentiation.

Advances in differentiation therapy of cancer are likely to depend on improved understanding of molecular events that underlie cell differentiation. We reported recently that cyclin-dependent kinase (Cdk)5 and p35Nck5a (p35) are expressed in human leukemia HL60 cells induced to differentiate to monocytes by an exposure to 1,25-dihydroxyvitamin D(3) (1,25D(3)), form a complex, and this complex has kinase activity (F. Chen and G. P. Studzinski, Blood 2001;97:3763). This laboratory has also provided evidence that the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is active in the early (24-48 h) stages of HL60 cell differentiation induced by 1,25D(3) but declines in the later, terminal phase of this form of differentiation (X. Wang and G. P. Studzinski, J Cell Biochem 2001;80:471). We examine now the hypothesis that Egr1 protein contributes to the up-regulation of p35 gene transcription and, thus, activated Cdk5/p35 kinase phosphorylates and inactivates mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1). Our data show that in 1,25D(3)-treated cells, p35 and Egr1 protein levels are elevated in a dose-dependent manner at the onset of the late stage of differentiation. We show also that 1,25D(3) treatment of HL60 cells markedly increases the binding of Egr1 to an element in the p35 gene promoter, whereas transfection of an excess of this Egr1-binding oligonucleotide ("promoter decoy") reduces p35 gene transcription and cell differentiation. Additionally, Cdk5/p35 phosphorylates MEK1 and inhibits its ability to phosphorylate its downstream target Erk2. These data suggest that in 1,25D(3)-treated HL60 cells, Egr1 up-regulates p35 gene transcription and that Cdk5/p35 kinase inactivates the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by phosphorylation of MEK1, and this contributes to terminal differentiation of these cells.

Control of phosphatase and tensin homolog (PTEN) gene expression in normal and neoplastic thyroid cells.

The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated. Cotransfection experiments indicated that in normal FRTL-5 rat thyroid cells, PTEN promoter activity was increased by overexpression of the transcription factor early growth response protein-1 (Egr-1). Moreover, Western blot experiments indicated that when Egr-1 expression was up-regulated by treating FRTL-5 cells with H2O2, an increase in PTEN expression was also observed. TSH induced opposite modifications on PTEN and Egr-1 protein levels. Moreover, acute or chronic TSH stimulation determined distinct effects. In fact, acute TSH stimulation (30 and 60 min) induced a decrease in PTEN, but an increase in Egr-1 protein levels. These effects were cAMP dependent; in fact, they were mimicked by forskolin. A chronic TSH treatment (5 d) stimulated PTEN protein expression, whereas Egr-1 protein was down-regulated. In contrast to normal thyroid cells, when the thyroid tumor cell lines ARO and BCPAP were exposed to H2O2, neither Egr-1 nor PTEN protein levels were increased. Acute stimulation of ARO and BCPAP cells with forskolin increased Egr-1, but not PTEN, protein levels. Therefore, thyroid tumor cell lines show alteration of PTEN gene expression regulation. RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr-1 mRNA is always paralleled by the absence of PTEN mRNA. Thus, modification of the Egr-1-dependent mechanisms may play a role in the silencing of PTEN gene expression occurring during thyroid cell transformation.

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6–6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.

The cAMP signaling system regulates LHbeta gene expression: roles of early growth response protein-1, SP1 and steroidogenic factor-1.

Expression of the gonadotropin genes has been shown to be modulated by pharmacological or physiological activators of both the protein kinase C (PKC) and the cAMP second messenger signaling pathways. Over the past few years, a substantial amount of progress has been made in the identification and characterization of the transcription factors and cognate cis-elements which mediate the PKC response in the LH beta-subunit (LHbeta) gene. In contrast, little is known regarding the molecular mechanisms which mediate cAMP-mediated regulation of this gene. Using pituitary cell lines, we now demonstrate that rat LHbeta gene promoter activity is stimulated following activation of the cAMP system by the adenylate cyclase activating agent, forskolin, or by the peptide, pituitary adenylate cyclase-activating peptide. The forskolin response was eliminated with mutation of a previously identified 3' cis-acting element for the early growth response protein-1 (Egr-1) when evaluated in the context of region -207/+5 of the LHbeta gene. Activation of the cAMP system increased Egr-1 gene promoter activity, Egr-1 protein levels and Egr-1 binding to the LHbeta gene promoter, supporting the role of this transcription factor in mediating the cAMP response. Analysis of a longer LHbeta promoter construct (-797/+5) revealed additional contribution by upstream Sp1 DNA-regulatory regions. Of interest, forskolin-induced stimulation of LHbeta gene promoter activity was observed to increase synergistically with introduction of the transcription factor, steroidogenic factor-1 (SF-1). Although SF-1 is a critical mediator of the cAMP response in other genes, mutation of the SF-1 DNA-binding sites in the rat LHbeta gene did not alter the forskolin response nor did forskolin increase SF-1 protein levels in a gonadotrope cell line. In a further set of experiments, it was determined that forskolin-responsiveness was maintained following mutation of the previously defined homeobox-binding element at position -100. We conclude that both Egr-1 and Sp1 contribute to cAMP-dependent transcription of the rat LHbeta gene promoter. While SF-1 does not act independently to mediate the cAMP/PKA response, SF-1 is important for magnification of this response.

Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma

BackgroundThe aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines.ResultsLow levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082). Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1–2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus.ConclusionsSuppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene.

Extracellular ATP stimulates the early growth response protein 1 (Egr-1) via a protein kinase C-dependent pathway in the human osteoblastic HOBIT cell line.

Extracellular nucleotides exert an important role in controlling cell physiology by activating intracellular signalling cascades. Osteoblast HOBIT cells express P2Y(1) and P2Y(2) G-protein-coupled receptors, and respond to extracellular ATP by increasing cytosolic calcium concentrations. Early growth response protein 1 (Egr-1) is a C(2)H(2)-zinc-finger-containing transcriptional regulator responsible for the activation of several genes involved in the control of cell proliferation and apoptosis, and is thought to have a central role in osteoblast biology. We show that ATP treatment of HOBIT cells increases Egr-1 protein levels and binding activity via a mechanism involving a Ca(2+)-independent protein kinase C isoform. Moreover, hypotonic stress and increased medium turbulence, by inducing ATP release, result in a similar effect on Egr-1. Increased levels of Egr-1 protein expression and activity are achieved at very early times after stimulation (5 min), possibly accounting for a rapid way for changing the osteoblast gene-expression profile. A target gene for Egr-1 that is fundamental in osteoblast physiology, COL1A2, is up-regulated by ATP stimulation of HOBIT cells in a timescale that is compatible with that of Egr-1 activation.

Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells.

Early growth response gene (Egr)-1 is a key transcription factor involved in vascular pathophysiology. Its role in diabetic vascular complications, however, remains unclear. Because hyperinsulinemia and hyperglycemia are major risk factors leading to diabetic vascular complications, we examined the effect of insulin and glucose on Egr-1 expression in murine glomerular vascular endothelial cells. Insulin or glucose, when added separately, increased egr-1 mRNA levels and promoter activity, as well as Egr-1 protein levels in nuclear extracts. When insulin was added to cells preincubated with glucose, the two had an additive effect on Egr-1 expression. Furthermore, vascular endothelial growth factor receptor-1 (flt-1) and plasminogen activator inhibitor-1, two known Egr-1-responsive genes, were also upregulated in the presence of insulin or glucose. An investigation into the underlying molecular mechanisms demonstrated that insulin, but not glucose, increased Egr-1 expression through extracellular signal-regulated kinase 1/2 activation, which is consistent with our previous reports. In contrast, inhibition of protein kinase C by phorbol ester or by the specific protein kinase C inhibitor chelerythrine chloride downregulated glucose-induced, but not insulin-induced, Egr-1 expression. Differential regulation of Egr-1 expression by insulin and glucose in vascular cells may be one of the initial key events that plays a crucial role in the development of diabetic vascular complications.

The egr- 1 gene is induced by DNA-damaging agents and non-genotoxic drugs in both normal and neoplastic human cells

The human egr- 1 gene encodes a zinc finger transcription factor induced by endogenous and exogenous stimuli such as growth factors, cytokines, and mitogens. Egr- 1 regulates other genes involved in growth and differentiation. The present study investigated the influence of genotoxic agents, such as chemotherapy drugs and other DNA damaging agents, on egr- 1 expression in normal and neoplastic cells. A transcriptional fusion between the human egr- 1 promoter and the enhanced green fluorescent protein ( EGFP) gene was used for direct visualization of intracellular Egr-1 regulation. The transcriptional activity of the egr- 1 promoter in this reporter system faithfully reflects intrinsic egr- 1 expression and induction, as demonstrated by FACS analysis of fluorescence and by RT–PCR for egr- 1. EGFP was expressed under the control of the egr- 1 promoter in stably transfected immortalized cell lines, such as HEK293, T98G, LNZ308, and 9L, which were then treated with genotoxic agents. A multitude of DNA damaging agents and therapeutic drugs caused significant upregulation of egr- 1 transcription. Furthermore, cytotoxic compounds without a direct DNA damaging effect, such as resveratrol and vincristine, which interfere with DNA replication and cell division, were also able to activate egr- 1 transcription. This suggests that cell cycle arrest rather than DNA damage seems to be the condition triggering egr- 1 transcription. Moreover, treatment with the MAP kinase (MAPK) inhibitor SB203580, which specifically blocks the stress inducible p38/SAPK2 pathway, did not alter egr- 1 induction. On the other hand, treatment with the inhibitor PD98059, which specifically blocks the MAPK/ERK pathway, partially suppressed the induction effect. In addition, the egr- 1 induction effect caused by genotoxic stress was found to be at least in part independent from the cellular p53 status, as it was observed in p53-deficient as well as in wild type p53 cell lines. These results suggest that induction of egr- 1, a gene to which until now no relation to DNA repair has been assigned, may belong to the fundamental cellular responses elicited by genotoxic and mitotic stress in normal as well as in neoplastic cells, and that enhanced levels of Egr-1 protein may be needed to regulate genes involved in DNA repair, cell survival, and apoptosis.

Increase of transcriptional levels of egr-1 and nur77 genes due to both nicotine treatment and withdrawal in pheochromocytoma cells.

The influence of nicotine on the expression of egr-1 and nur77 genes by nicotine treatment and withdrawal was assessed using PC12 cells. Nicotine treatment significantly increased the amount of mRNA for egr-1 and nur77 genes at 0.5 h post-nicotine treatment in the PC12 cells. In addition, nicotine withdrawal also elevated transcriptional levels of egr-1 and nur77 genes in Northern blot analyses. Nicotine treatment (200 microM) was also found to significantly increase expressional levels of Egr-1 and Nur77 proteins at 0.5 h post-nicotine treatment. In contrast, Egr-1 and Nur77 protein levels were dramatically decreased by nicotine withdrawal. These results suggest that expressional levels of Egr-1 and Nur77 proteins in neural cells may affect the transcriptional activity of late-response genes after nicotine withdrawal.

Early Growth Response Gene-1 Regulates the Expression of the Rat Luteinizing Hormone Receptor Gene1

LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between -171 and -137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between -171 and -137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.

The extracellular signal-regulated protein kinases Erk1/Erk2 stimulate expression and biological activity of the transcriptional regulator Egr-1.

The zinc finger protein early growth response 1 (Egr-1) is a transcriptional activator involved in the regulation of growth and differentiation. We show here that a constitutive active mutant of mitogen-activated kinase kinase-1 (MAPKK-1) strongly stimulates the activity of the Egr-1 promoter, thus explaining the effects of mitogens upon Egr-1 mRNA and protein levels. Moreover, we show that a constitutive active MAPKK-1 leads to an increase in the biological activity of Egr-1 to activate transcription. We conclude that the signaling pathway involving mitogen-activated protein kinase/extracellular signal-regulated protein kinase has a dual impact on the biology of Egr-1 by controlling the transcription of the Egr-1 gene and the transcriptional activity of the Egr-1 protein.

Elevated Egr-1 in Human Atherosclerotic Cells Transcriptionally Represses the Transforming Growth Factor-β Type II Receptor

Atherosclerotic lesions may progress due to a "failure to die" by vascular repair cells. Egr-1, a zinc finger transcription factor, is elevated more than 5-fold in human carotid lesions relative to the adjacent tunica media. Lesion cells in vitro also express 2-3-fold higher Egr-1 mRNA and protein levels but express much lower levels of the transforming growth factor-beta (TGF-beta) Type II receptor (TbetaR-2) and are functionally resistant to the antiproliferative effects of TGF-beta. Lesion cells fail to express a TbetaR-2 promoter/chloramphenicol acetyltransferase (CAT) construct but overexpress an Egr-1-inducible platelet-derived growth factor-A promoter/CAT construct. Transfection of Egr-1 cDNA represses TbetaR-2/CAT constructs but induces PDGF-A/CAT. Egr-1 transfection reduces the levels of TbetaR-2 and confers resistance to the antiproliferative effect of TGF-beta1. Egr-1 can interact directly with both the -143 Sp1 site and the positive regulatory element 2 (PRE2) (ERT/ets) region of the TbetaR-2 promoter. Thus, although activating a family of stress-responsive genes, Egr-1 also transcriptionally represses one of the major inhibitory pathways that restrains vascular repair.

Egr-1 gene is induced by the systemic administration of the vascular endothelial growth factor and the epidermal growth factor

Egr-1 is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. In cultured endothelial cells, the Egr-1 gene has been shown to respond to a variety of extracellular signals. However, the physiological relevance of these findings remains unclear. To address this question, the growth factor-mediated response of the Egr-1 gene under in vivo conditions was analyzed. To that end, either vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) was injected into the intraperitoneal cavity of mice. Growth factors were delivered to all tissues examined, as evidenced by the widespread distribution of I125-labeled growth factors and the phosphorylation of their respective receptors. In Western blot analyses of whole-tissue extracts, Egr-1 protein levels were shown to be induced in the heart, brain, liver, and spleen of VEGF-treated mice, and in the heart, lung, brain, liver and skeletal muscle of EGF-treated animals. Changes in Egr-1 levels did not correlate with changes in receptor phosphorylation or ERK1/2 phosphorylation. In Northern blot analyses, VEGF induced Egr-1 mRNA levels in all tissues examined except lung and kidney, whereas EGF led to increased transcripts in all tissues except kidney. In immunofluorescence studies, VEGF induced Egr-1 in microvascular endothelial cells of the heart and liver, and EGF induced Egr-1 in the microvascular bed of skeletal muscle. Taken together, these results suggest that the Egr-1 gene is differentially regulated in response to systemically administered VEGF and EGF.

Egr-1 gene is induced by the systemic administration of the vascular endothelial growth factor and the epidermal growth factor

AbstractEgr-1 is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. In cultured endothelial cells, the Egr-1 gene has been shown to respond to a variety of extracellular signals. However, the physiological relevance of these findings remains unclear. To address this question, the growth factor-mediated response of the Egr-1 gene under in vivo conditions was analyzed. To that end, either vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) was injected into the intraperitoneal cavity of mice. Growth factors were delivered to all tissues examined, as evidenced by the widespread distribution of I125-labeled growth factors and the phosphorylation of their respective receptors. In Western blot analyses of whole-tissue extracts, Egr-1 protein levels were shown to be induced in the heart, brain, liver, and spleen of VEGF-treated mice, and in the heart, lung, brain, liver and skeletal muscle of EGF-treated animals. Changes in Egr-1 levels did not correlate with changes in receptor phosphorylation or ERK1/2 phosphorylation. In Northern blot analyses, VEGF induced Egr-1 mRNA levels in all tissues examined except lung and kidney, whereas EGF led to increased transcripts in all tissues except kidney. In immunofluorescence studies, VEGF induced Egr-1 in microvascular endothelial cells of the heart and liver, and EGF induced Egr-1 in the microvascular bed of skeletal muscle. Taken together, these results suggest that the Egr-1 gene is differentially regulated in response to systemically administered VEGF and EGF.

Cyclical expression of egr-1/NGFI-A in the rat anterior pituitary: a molecular signal for ovulation?

Molecular genetic studies of egr-1/NGFI-A have recently established a key role for this immediate early gene in anterior pituitary development, and particularly in transcriptional regulation of the LH beta-subunit gene. These studies, using null mutant mice, addressed the developmental aspects of gonadotrophin gene regulation by egr-1 and, because of the limitations of this approach, did not address the role of egr-1 in adult reproductive physiology. Using the rat, we have now investigated the expression of egr-1 during the female reproductive cycle, and have found evidence of cyclical changes in anterior pituitary egr-1 expression, at mRNA, protein, and DNA binding activity levels. Specifically, we have shown that egr-1 mRNA is significantly elevated during proestrous, and conversely suppressed on the subsequent day of oestrous. We have also demonstrated significantly raised levels of an approximately 70 kDa Egr-1 immunoreactive protein band during the night of proestrous, together with markedly raised levels of a Egr-1 consensus sequence DNA binding complex. These studies are indicative of both direct egr-1 gene regulation by oestrogen, and also regulation by GnRH. Finally, we have sequenced previously uncharacterized regions of the egr-1 gene and found evidence of a potential oestrogen response element. Our findings are consistent with the hypothesis that Egr-1 forms at least part of the molecular signal for ovulation in the pituitary.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Sp1. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-1 DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP.

Phorbol ester activation of the neuronal nicotinic acetylcholine receptor alpha7 subunit gene: involvement of transcription factor Egr-1.

alpha-Bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells are up-regulated by long-term exposure to phorbol esters. The rise in receptor density is paralleled by an increase in transcripts corresponding to the alpha7 subunit, which is a component of this receptor subtype. Transcriptional activation of the alpha7 subunit gene is evidenced in reporter gene transfection experiments, in which phorbol esters increase alpha7 promoter activity by up to 14-fold. About 80% of this activation is abolished when at least two of the three sites for the immediate-early transcription factor Egr-1, present in the proximal promoter region of the alpha7 subunit gene, are mutated simultaneously. In addition, phorbol esters elevate both Egr-1 mRNA and Egr-1 protein levels in chromaffin cells, whereas electrophoretic mobility shift assays show that the Egr-1 component of the complexes that originate at the alpha7 promoter increases in cells treated with phorbol esters. These results suggest that the transcription factor Egr-1 is involved in triggering expression of alpha-bungarotoxin-sensitive nicotinic receptors in response to external stimuli, such as the ones resulting from phorbol ester treatment, and support our previous hypothesis that the alpha7 subunit gene is one of the specific targets for Egr-1.

Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor EGR-1

Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor Egr-1. In previous studies, we found a close link of early growth response gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antiproliferative agents inhibited mitogen-induced Egr-1 expression. Here we investigated the effect of S-nitrosoglutathione (GSNO) on the proliferation of MCs, specifically asking how GSNO regulates the transcription factor Egr-1, which we have previously shown to be critical for the induction of MC mitogenesis. The proliferation of MCs was measured by thymidine incorporation and cell counting. Egr-1 mRNA and protein levels were detected by Northern and Western blots. Electrophoretic mobility shift assays (EMSAs) and chloramphenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1. GSNO strongly inhibited serum-induced MC proliferation (-84% at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GSNO concentrations from 50 to 200 micromol/L, whereas mRNA levels increased again at concentrations above 500 micromol/L. This increased mRNA expression, however, was not translated into Egr-1 protein. Instead, Egr-1 protein induction was inhibited (-40%). EMSAs indicated that GSNO inhibited specific binding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional activation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO. Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This antimitogenic property is mediated at least in part by inhibitory effects of GSNO on Egr-1 protein levels and by reducing the ability of Egr-1 to activate transcription by impairing its DNA binding activity.

Cholinergic stimulation of early growth response-1 DNA binding activity requires protein kinase C and mitogen-activated protein kinase kinase activation and is inhibited by sodium valproate in SH-SY5Y cells.

Activation of muscarinic receptors in human neuroblastoma SH-SY5Y cells with carbachol stimulated a rapid and large increase in early growth response-1 (Egr-1, also called zif268 and NGF1-A) protein levels and DNA binding activity. Egr-1 DNA binding activity was stimulated within 15 min of treatment with carbachol and maintained a maximum 20-fold increase over basal between 1 and 2 h after treatment, and the EC50 was approximately 1 microM carbachol. Carbachol-stimulated Egr-1 DNA binding activity was dependent on protein kinase C, as it was potently inhibited by GF109203X (IC50 approximately 0.1 microM) and was reduced by 85 +/- 5% by down-regulation of protein kinase C. Inhibitors of increases in intracellular calcium levels reduced carbachol-induced Egr-1 DNA binding activity by 25-35%. Carbachol-stimulated activation of Egr-1 was reduced 35% by genistein, a tyrosine kinase inhibitor, and 60% by PD098059, an inhibitor of mitogen-activated protein kinase kinases 1/2 (MEK1/2) that activates extracellular-regulated kinases 1/2 (ERK1/2). A novel inhibitory action was caused by chronic (7-day) administration of sodium valproate but not by two other bipolar disorder therapeutic agents, lithium and carbamazepine. Valproate treatment reduced carbachol-stimulated Egr-1 DNA binding activity by 60% but did not alter carbachol-induced activation of ERK1/2 or p38 or increases in Egr-1 protein levels. These results reveal that muscarinic receptors activate Egr-1 through a signaling cascade primarily encompassing protein kinase C, MEK1/2, and ERK1/2 and that valproate substantially inhibits Egr-1 DNA binding activity stimulated by carbachol or protein kinase C.