Expression Of Early Growth Response Gene-1 Research Articles

EGR1 Predicts PTEN and Survival in Patients With Non–Small-Cell Lung Cancer

The zinc finger transcription factor early growth response gene 1 (EGR1) is underexpressed in non-small-cell lung cancer (NSCLC) compared with normal lung. EGR1 expression has been linked to tumor suppression as a result of cell cycle arrest and apoptosis through regulation of tumor suppressor pathways including PTEN. For these reasons, we hypothesized that reduced levels of EGR1 would correlate with inferior outcome in patients with NSCLC. Patients who underwent surgical resection for NSCLC had RNA extracted from tumor tissue and EGR1 gene expression was quantified by real-time quantitative polymerase chain reaction. The levels of EGR1 expression were examined in relationship to patient characteristics, histology, tumor stage, PTEN expression, and overall and disease-free survival. EGR1 expression strongly correlated with PTEN expression (P < .0001). No correlation of EGR1 with histology or stage was detected. Patients with high levels of EGR1 had better overall and disease-free survival compared with patients with low levels of EGR1 (P = .040 and P = .096, respectively). In a stratified log-rank test, low EGR1 expression was predictive of poor survival independent of tumor stage. EGR1 gene expression predicts PTEN levels and survival after surgical resection of NSCLC. Consistent with its known tumor suppressor properties, lower levels of EGR1 are associated with poor outcome. Identification of patients with low EGR1 therefore may identify patients at high risk for disease recurrence and may also identify patients who have tumors resistant to therapy secondary to loss of pathways such as PTEN.

Insulin and Oxidant Stress Effects on Early Growth Response Gene-1 Expression are Mediated Differently in Vascular Smooth Muscle Cells

Insulin and Oxidant Stress Effects on Early Growth Response Gene-1 Expression are Mediated Differently in Vascular Smooth Muscle Cells

Early Growth Response Gene 1 Modulates Androgen Receptor Signaling in Prostate Carcinoma Cells

The transcription factor early growth response gene 1 (EGR1) has been implicated in diverse roles in the regulation of cell growth, apoptosis, and differentiation. Previous studies suggest that the effects of EGR1 on tumorigenesis are critically dependent on the cellular context. In a majority of prostate cancers, EGR1 is overexpressed and promotes prostate tumor progression. In contrast, in other tumor types such as breast cancers and glioblastomas, EGR1 is expressed at low levels and when overexpressed can inhibit tumor growth. To explore the role of EGR1 in prostate tumorigenesis, we examined the impact of EGR1 expression on the androgen receptor (AR) signaling pathway. We show here that EGR1 binds to the AR in prostate carcinoma cells, and an EGR1-AR complex can be detected by chromatin immunoprecipitation at the enhancer of an endogenous AR target gene. Overexpression of EGR1 enhanced AR-mediated transactivation, whereas EGR1 knockdown by small interfering RNA inhibited AR signaling pathway activity. Furthermore, Western blot and immunocytochemical analyses showed that constitutive overexpression of EGR1 promotes the translocation of AR from the cytoplasm to the nucleus. These results indicate that EGR1 may promote prostate cancer development by modulating the androgen receptor signaling pathway.

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.

Acute intrarenal infusion of ANG II does not stimulate immediate early gene expression in the kidney.

ANG II is capable of stimulating expression of immediate early genes such as egr-1 and c-fos in a variety of cultured cells, including cells of renal origin. To investigate whether ANG II can stimulate early growth response gene expression in vivo, we studied the effects of acute renal artery infusion of low-dose ANG II (2.5 ng small middle dot kg(-1) small middle dot min(-1)) or vehicle on the renal expression of c-fos and egr-1 genes in rats. ANG II infusion for 30 or 240 min decreased renal vascular conductance by approximately 13 and 8%, respectively, compared with the vehicle group. Expression of the early growth response genes c-fos and egr-1 was analyzed using Northern blot hybridization. No significant upregulation of c-fos or egr-1 mRNA levels was detected in rats that received ANG II for either 30 or 240 min, compared with the vehicle groups. We conclude that ANG II, at doses that cause significant physiological effects, does not increase the renal expression of c-fos or egr-1 genes over periods of up to 4 h in vivo.

EGR-1, a UV-Inducible Gene in p53−/− Mouse Cells

Changes in gene expression were examined in p53−/− and p53+/+ mouse cells after ultraviolet (UV) irradiation. Differential display was used to identify differentially expressed gene(s) in UV-treated p53−/− and p53+/+ cells. One of the differentially expressed genes was EGR-1 (early growth response gene-1), which was shown to be induced only in p53−/− cells. The induction of this gene by UV was detected as early as 0.5 h, peaked at 2 h, and returned to normal levels by 4 h. De novo protein synthesis was not required for UV-induced EGR-1 expression in p53−/− cells. Pretreatment of p53−/− cells with suramin, an inhibitor of growth factor receptors, completely suppressed UV-induced EGR-1 expression, suggesting that the induction may be mediated via the growth factor receptors. The presence of wild-type p53 suppressed the induction of EGR-1 after UV treatment. Overexpression of EGR-1 promoted the UV-induced transformation in p53+/+ cells, but not in p53−/− cells. These data suggested that EGR-1 may be an important player in the UV responses of mammalian cells and may influence UV-induced transformation.

Induction of Early Growth Response-1 Gene Expression by Calmodulin Antagonist Trifluoperazine through the Activation of Elk-1 in Human Fibrosarcoma HT1080 Cells

The early growth response gene-1 (Egr-1) is a transcription factor that plays an important role in cell growth and differentiation. It has been known that Egr-1 expression is down-regulated in many types of tumor tissues, including human fibrosarcoma HT1080 cells, and introduction of the Egr-1 gene into HT1080 cells inhibits cell growth and tumorigenic potential. Trifluoperazine (TFP), a phenothiazine class calmodulin antagonist, is known to inhibit DNA synthesis and cell proliferation and potentially important in antitumor activities. To understand the regulatory mechanism of Egr-1, we investigated the effect of TFP on expression of Egr-1 in HT1080 cells. Herein, we report that Egr-1 expression was increased by TFP in synergy with serum at the transcriptional level. Both the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN62 and the calcineurin inhibitor cyclosporin A enhanced TFP-dependent increase of Egr-1, suggesting that the Ca(2+)/calmodulindependent pathway plays a role in regulation of Egr-1 expression in HT1080 cells. The TFP-stimulated increase of the Egr-1 protein was preferentially inhibited by the MEK-specific inhibitor PD98059. In addition, activation of human Egr-1 promoter and the transcriptional activation of the ternary complex factor Elk-1 induced by TFP were inhibited both by pretreatment of PD98059 and by expression of the dominant-negative RasN17. These results indicate that the Ras/MEK/Erk/Elk-1 pathway is necessary for TFP-induced Egr-1 expression. We propose that the calmodulin antagonist TFP stimulates Egr-1 gene expression by modulating Ras/MEK/Erk and activation of the Elk-1 pathway in human fibrosarcoma HT1080 cells.

Hypoxia-associated Induction of Early Growth Response-1 Gene Expression

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.

Modulation of Ras/Raf/Extracellular Signal–Regulated Kinase Pathway by Reactive Oxygen Species Is Involved in Cyclic Strain–Induced Early Growth Response-1 Gene Expression in Endothelial Cells

Endothelial cells (ECs) exposed to cyclic strain induce gene expression. To elucidate the signaling mechanisms involved, we studied the effects of cyclic strain on ECs by using early growth response-1 (Egr-1) as a target gene. Cyclic strain induced a transient increase of Egr-1 mRNA levels that resulted in an increase of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. ECs subjected to strain enhanced Egr-1 transcription as revealed by promoter activities. Catalase pretreatment inhibited this induction. ECs, transfected with a dominant positive mutant of Ras (RasL61), increased Egr-1 promoter activities. In contrast, transfection with a dominant negative mutant of Ras (RasN17) attenuated this strain inducibility. ECs transfected with a dominant negative mutant of Raf-1 (Raf301) or the catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK2) diminished strain-induced promoter activities. However, little effect on strain inducibility was observed in ECs transfected with a dominant negative mutant of Rac (RacN17) or a catalytically inactive mutant of JNK (JNK[K-R]). Consistently, strain-induced Egr-1 expression was inhibited after ECs were treated with a specific inhibitor (PD98059) to mitogen-activated protein kinase kinase. Moreover, strain to ECs induced mitogen-activated protein kinase/ERK activity. The activation of the ERK pathway was further substantiated by an increase of strain-induced transcriptional activity of Elk1, an ERK substrate. This strain-induced ERK activity was attenuated after ECs were treated with N-acetylcysteine or catalase. Consequently, this Egr-1 gene induction was abolished after ECs were treated with N-acetylcysteine or catalase. Deletion analyses of the promoter region (-698 bp) indicated that cyclic strain and H2O2 shared a common serum response element. Our data clearly indicate that cyclic strain-induced Egr-1 expression is mediated mainly via the Ras/Raf-1/ERK pathway and that strain-induced reactive oxygen species can modulate Egr-1 expression at least partially via this signaling pathway.

In situ expression of the early growth response gene-1 during murine nephrogenesis

WT1 maps to chromosome 11p13 and encodes a deoxyribonucleic acid (DNA) binding protein whose expression is necessary for normal urogenital development. The WT1 protein binds to some of the same DNA sequences as the early growth response gene-1 (EGR-1) protein, the latter being an immediate-early gene product that activates or represses transcription in a promoter and cell-specific manner. Transient transfection experiments have shown that WT1 can repress EGR-1 activated transcription from the EGR-1 promoter. To determine if WT1 is likely to be a physiologically important repressor of EGR-1 we performed ribonucleic acid (RNA) in situ hybridization of EGR-1 on sequential sagittal sections of murine embryos before and throughout nephrogenesis, and compared the results to our previous study of WT1 expression during murine embryogenesis. Prior to embryological day 9.5 WT1 messenger RNA expression is absent in the embryo proper but is expressed in the maternal uterus. With the initiation of organogenesis on embryological day 10.5 WT1 messenger RNA localizes within the pronephric and mesonephric tissues. By embryological day 11.5 the nephrogenic cord, urogenital ridge and metanephric tissue have WT1 hybridization signals and increasingly centripetal expression of WT1 in the kidney correlates with differentiation from embryological days 11.5 to 16.5. In contrast to previous reports of the tissue restricted expression of WT1, EGR-1 expression by in situ hybridization was apparent in all 3 germ layers and their derivatives throughout embryogenesis. Down-regulation of EGR-1 expression occurred in the maternal uterus as well as the metanephric blastema and its derivatives during renal development. This observation defines a spatial and temporal window during which WT1 competition for EGR-1 DNA binding sites may be involved in regulating EGR-1 expression.

Activation of pp90 rsk and early growth response-1 gene expression by pokeweed mitogen in human B cells

The present studies have examined the effects of pokeweed mitogen (PWM) on the induction of early growth response-1 gene ( EGR-1) in normal human B cells. PWM regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the chloramphenicol acetyltransferase ( CAT) gene demonstrated that PWM induced EGR-1 transcription is conferred by the CArG motif (C C[AT] 6GG) in the EGR-1 promoter. The results further demonstrated the activation of S6 kinase (pp90 rsk), evidenced by phosphorylation of S6 and serum response factor (SRF) peptides, in PWM treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells.

Constitutive and Inducible Levels of egr-1 and c-myc Early Growth Response Gene Expression in Self-Renewing B-1 Lymphocytes

B-1 lymphocytes constitute a mature B cell population that differs from conventional B cells in signaling requirements for cell cycle progression, being unusually responsive to PKC agonism but refractory to antigen receptor stimulation. Further, B-1 cells are self-renewing and derive from surface immunoglobulin-positive precursors. A previous report on clonally derived B-1 cells suggested that increased levels of expression of c-myc might underlie these unusual growth characteristics. This issue has now been reexamined using primary B-1 cells. The possible role of egr-1, in addition to c-myc, in specifying the responsiveness of B-1 cells was evaluated by determining levels of gene expression at baseline and after mitogenic treatment. Expression of the two genes was similar in B-1 and B-2 cells prior to stimulation. Subsequently, B-1 cells responded with higher levels of gene expression than B-2 cells regardless of ultimate cell cycle progression, with the exception of stimulation by anti-Ig. Overall, there was no apparent direct correlation between stimulated levels of expression of egr-1 or c-myc and mitogen-induced S phase entry, nor were B-1 cells characterized by constitutively elevated levels of either proto-oncogene that might explain their capacity for self-renewal.

Activation of serine/threonine protein kinases and early growth response 1 gene expression by tumor necrosis factor in human myeloid leukemia cells.

The early growth response 1 (EGR-1) gene is induced by mitogenic and differentiating signals in diverse cell types. The present studies have examined the effects of TNF-alpha on the induction of EGR-1 expression in human myeloid leukemia cells and the potential cytoplasmic signaling cascades that transduce TNF-induced signals to the nucleus. The results demonstrate that treatment of HL-60 cells with TNF is associated with the transient induction of the EGR-1 gene. The results also demonstrate that TNF treatment is associated with activation of the serine/threonine kinase, pp90rsk, which acts upstream to EGR-1 gene induction. Partial purification of pp90rsk by affinity chromatography demonstrated an increase in S6 peptide phosphorylation in response to TNF treatment. Because TNF activates sphingomyelin hydrolysis, we also studied the effects of sphingomyelinase (SMase) on induction of EGR-1 and pp90rsk. The results demonstrate that SMase also activates pp90rsk and induces EGR-1 gene expression. Previous work has demonstrated that mitogen-activated protein (MAP) kinase activates pp90rsk. The present studies further show that treatment with TNF or SMase is associated with induction of both the pp42/44 MAP and the related Jun kinases. Induction of pp42/44 MAP kinase activity is temporally related to activation of pp90rsk and the EGR-1 gene. These findings support the involvement of an MAP kinase/pp90rsk/EGR-1 cascade in the response of myeloid leukemia cells to TNF.

Nitric oxide reduces early growth response-1 gene expression in rat lung macrophages treated with interferon-gamma and lipopolysaccharide.

Since early growth response-1 (Egr-1) is required for macrophage differentiation and nitric oxide (NO) is immunosuppressive, we hypothesized that NO would reduce Egr-1 expression in rat lung macrophages. The inflammatory stimuli interferon-gamma and lipopolysaccharide induced an early, transient increase in Egr-1 mRNA (> 5-fold at 2 h) and a sustained, high level of inducible NO synthase mRNA (> 100-fold from 4 to 24 h). The NO metabolites nitrite and nitrate rose > 10-fold in medium from stimulated versus unstimulated cells over 24 h. Concomitant with elevated nitrogen oxides, Egr-1 mRNA levels declined to 80% below unstimulated cells at 24 h. This decline was blocked by an inhibitor of NO production, NG-monomethyl-L-arginine. Further, the NO donor S-nitroso-N-acetylpenicillamine inhibited Egr-1 expression in a dose-dependent manner, producing complete inhibition at 0.5 mM. The effect of S-nitroso-N-acetylpenicillamine was not due to reduced macrophage viability. We conclude that Egr-1 induction precedes inducible NO synthase induction in stimulated rat macrophages and that subsequent NO production reduces macrophage expression of Egr-1. We propose that this mechanism is used to regulate macrophage differentiation in human immunodeficiency virus infection and other inflammatory states.

Action of dihydropyridine calcium antagonists on early growth response gene expression and cell growth in vascular smooth muscle cells

Evidence suggests that calcium antagonists may suppress vascular smooth muscle cell (VSMC) growth and proliferation, which may be a crucial step in the pathogenesis of hypertension and atherosclerosis. The effects of the dihydropyridine calcium antagonists nifedipine, nitrendipine, nisoldipine, nimodipine and isradipine on cell growth induced by platelet-derived growth factor (PDGF)-AB and angiotensin II (Ang II), and expression of the transcription factors c-fos and early-growth response gene 1 (egr-1) were investigated. Proliferation of VSMC in culture was measured by [3H]-thymidine incorporation into cell DNA and by cell count. Expression of c-fos and egr-1 messenger RNA (mRNA) was determined by the Northern blot technique. All of the calcium antagonists blunted the PDGF-induced rise in VSMC DNA synthesis. The inhibitory potency of isradipine on PDGF-stimulated DNA synthesis was approximately 10-fold that of the other calcium antagonists used, isradipine having a half-maximal inhibitory concentration (IC50) of (4.2 +/- 0.16) x 10(-7) mol/l. The calcium antagonists investigated also inhibited Ang II-induced DNA synthesis. Isradipine (10(-6) mol/l) completely abolished the PDGF-induced cell proliferation. Both PDGF (50 ng/ml) and Ang II (10(-7) mol/l) induced c-fos and egr-1 mRNA expression, having maximum effect after 30 min. In the case of c-fos, pre-incubation with 5 x 10(-6) mol/l isradipine led to a decrease in both Ang II- and PDGF-induced expression of this immediate-early gene. The expression of egr-1 was not affected by pre-incubation with 5 x 10(-6) mol/l isradipine. All calcium antagonists investigated in the present study inhibited cell growth. Isradipine was more potent in blocking growth factor-induced cell growth than the other calcium antagonists studied. The inhibitory effect of the dihydropyridine calcium antagonists appears to be dependent on the expression of c-fos.

Dimethyl sulfoxide inhibits the expression of early growth-response genes and arrests fibroblasts at quiescence

We have previously shown that dimethyl sulfoxide (DMSO) treatment of mouse embryo fibroblasts (MEF) at the early hours of mitogenic stimuli resulted in the inhibition of DNA and protein synthesis; delayed treatment of serum-stimulated cells with DMSO had little effect on the synthesis of these macromolecules. Here, we demonstrate the specific inhibition of expression of early growth response genes by DMSO in serum-stimulated MEF. The expression of interleukin 6 and of oncogenes c- myc and c- fos were inhibited when the cells were treated with 2% DMSO from the beginning of serum-stimulated growth but not after 3 h of mitogenic stimuli. Although the actin gene is an early serum-response gene, its expression was not affected by DMSO. The synthesis of another serum-induced protein, the plasminogen activator inhibitor-1 was blocked during concurrent and delayed (after 3 h of stimulation) treatment of serum-stimulated fibroblasts with DMSO. The expression of glyceraldehyde-3-phosphate dehydrogenase gene was not affected by DMSO. These results indicate that the expression of non-growth-related genes are either not affected or affected nonspecifically both at early and late stages of serum-induced growth of mouse embryo fibroblasts. The serum-induced expression of c- fos gene was abolished by DMSO treatment of MEF while the phorbol 12-myristate 13-acetate-induced expression of fos gene was not, indicating that the PMA signaling pathway was refractory to DMSO. Treatment of cells with medium containing 2% DMSO for 24–48 h prevents them from progression into cell cycle by preventing the expression of genes involved in G 0–G 1 transition of quiescent cells.

PKC activity and early growth response gene expression during diethylnitrosamie rat hepatocarcinogenesis

PKC activity and early growth response gene expression during diethylnitrosamie rat hepatocarcinogenesis