Egr-1 mRNA Expression Research Articles

A Novel Role of Brg1 in the Regulation of SRF/MRTFA-dependent Smooth Muscle-specific Gene Expression

Serum response factor (SRF) is a key regulator of smooth muscle differentiation, proliferation, and migration. Myocardin-related transcription factor A (MRTFA) is a co-activator of SRF that can induce expression of SRF-dependent, smooth muscle-specific genes and actin/Rho-dependent genes, but not MAPK-regulated growth response genes. How MRTFA and SRF discriminate between these sets of target genes is still unclear. We hypothesized that SWI/SNF ATP-dependent chromatin remodeling complexes, containing Brahma-related gene 1 (Brg1) or Brahma (Brm), may play a role in this process. Results from Western blotting and qRT-PCR analysis demonstrated that dominant negative Brg1 blocked the ability of MRTFA to induce expression of smooth muscle-specific genes, but not actin/Rho-dependent early response genes, in fibroblasts. In addition, dominant negative Brg1 attenuated expression of smooth muscle-specific genes in primary cultures of smooth muscle cells. MRTFA overexpression did not induce expression of smooth muscle-specific genes in SW13 cells, which lack endogenous Brg1 or Brm. Reintroduction of Brg1 or Brm into SW13 cells restored their responsiveness to MRTFA. Immunoprecipitation assays revealed that Brg1, SRF, and MRTFA form a complex in vivo, and Brg1 directly binds MRTFA, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated the ability of MRTFA to increase SRF binding to the promoters of smooth muscle-specific genes, but not early response genes. Together these data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in regulating expression of SRF/MRTFA-dependent smooth muscle-specific genes but not SRF/MRTFA-dependent early response genes.

EARLY GROWTH RESPONSE 1 MEDIATES THE SYSTEMIC AND HEPATIC INFLAMMATORY RESPONSE INITIATED BY HEMORRHAGIC SHOCK

Hemorrhagic shock (HS) is a major cause of morbidity and mortality in trauma patients. The early growth response 1 (Egr-1) transcription factor is induced by a variety of cellular stresses, including hypoxia, and may function as a master switch to trigger the expression of numerous key inflammatory mediators. We hypothesized that HS would induce hepatic expression of Egr-1 and that Egr-1 upregulates the inflammatory response after HS. The Egr-1 mice and wild-type (WT) controls (n>or=5 for all groups) were subjected to HS alone or HS followed by resuscitation (HS/R). Other mice were subjected to a sham procedure which included general anesthesia and vessel cannulation but no shock (sham). After the HS, HS/R, or sham procedures, mice were euthanized for determination of serum concentrations of interleukin (IL) 6, IL-10, and alanine aminotransferase. Northern blot analysis was performed to evaluate Egr-1 messenger RNA (mRNA) expression. Liver whole cell lysates were evaluated for Egr-1 protein expression by Western blot analysis. Hepatic expression of IL-6, granulocyte colony-stimulating factor, and intracellular adhesion molecule 1 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The Egr-1 DNA binding was assessed using the electrophoretic mobility shift assay. Hemorrhagic shock results in a rapid and transient hepatic expression of Egr-1 mRNA in WT mice by 1 h, whereas protein and DNA binding activity was evident by 2.5 h. The Egr-1 mRNA expression diminished after 4 h of resuscitation, whereas Egr-1 protein expression and DNA binding activity persisted through resuscitation. The Egr-1 mice exhibited decreased levels of hepatic inflammatory mediators compared with WT controls with a decrease in hepatic mRNA levels of IL-6 by 42%, granulocyte colony-stimulating factor by 39%, and intracellular adhesion molecule 1 by 43%. Similarly, Egr-1 mice demonstrated a decreased systemic inflammatory response and hepatic injury after HS/R compared with their WT counterparts. Early growth response 1 is rapidly upregulated in the liver during and after resuscitation from HS. Our results showing a blunted inflammatory response in Egr-1 mice provides evidence that Egr-1 functions as a proximal signal transduction mechanism responding to shock by amplifying the systemic inflammatory response.

Egr‐1 transcription activation exists in placental endothelium when vascular disease is present

To seek evidence of early vascular injury in the placental villous microcirculation in placental insufficiency identified by a high-resistance umbilical Doppler study by examining for expression of fibroblast growth factor receptor-1 (FGFR-1), its transcription factor, early growth response factor-1 (Egr-1) and plasma fibroblast growth factor-2 (FGF-2). Case-control study. University teaching hospital. Placentas and umbilical vein blood were collected at delivery from 12 women with normal pregnancy delivered at term and 14 with placental vascular disease defined by an abnormal umbilical artery Doppler study. Microvascular endothelial cells were isolated from fresh human placentas using collagenase digestion and Dynabeads coated with monoclonal antibody against CD31. RNA was extracted from the isolated endothelial cells. The messenger RNA (mRNA) expression of FGFR-1 and Egr-1 production were assessed by reverse transcription polymerase chain reaction and factored relative to 18S ribosomal RNA. To confirm that FGF-2 was playing a significant role in this microvascular endothelial cell injury in the placenta, we also measured the soluble fraction of FGF-2 in fetal plasma from same groups of pregnancies using an enzyme-linked immunosorbent assay. Microvascular endothelial cells expression of Egr-1mRNA, FGFR-1 mRNA and presence of soluble FGF-2 in fetal plasma. The soluble level of FGF-2 in the fetal placental circulation from pregnancy with placental vascular disease was increased when compared with normal pregnancy (median 10.15 pg/ml and interquartile range 5.34-21.83 pg/ml versus 4.46 pg/ml and 3.69-5.66 pg/ml; P < 0.05). Microvascular endothelial cells from the placental villi with placental vascular disease showed upregulation of both FGFR-1 mRNA expression (median 0.72 and interquartile range 0.40-1.64 versus 0.34 and 0.19-0.71; P<0.05) and Egr-1 expression (median 0.79 and interquartile range 0.27-1.86 versus 0.23 and 0.17-0.67; P<0.05) in comparison with normal pregnancy. Endothelial cells from the placental villi are upregulated for expression of Egr-1 transcription factor gene in placental vascular disease. The FGFR-1 activation and increase in FGF-2 in the fetal circulation are known to be very early features of the response of endothelium to injury. Egr-1 is a promoter of many key pathophysiologically relevant target genes, which influence the development of subsequent vascular lesions. This change may occur before the pathological features recognised on microscopy.

De novo synthesis of early growth response factor-1 is required for the full responsiveness of mast cells to produce TNF and IL-13 by IgE and antigen stimulation

AbstractEarly growth-response factor 1 (Egr-1) is a zinc-finger transcription factor that plays a regulatory role in the expression of many genes important for inflammation. Whether Egr-1 is involved in IgE-dependent mast-cell activation was investigated. We demonstrated that IgE and antigen (TNP) stimulation induced a rapid expression of Egr-1 mRNA in mouse bone marrow–derived mast cells (BMMCs). As early as 15 to 20 minutes after IgE + TNP stimulation, Egr-1 protein was detectable in the nucleus of BMMCs by immunofluorescence or electrophoretic mobility shift assay. To examine a role for Egr-1 in IgE-dependent cytokine production by mast cells, Egr-1–deficient (Egr-1–/–) BMMCs were developed from the bone marrow cells of Egr-1 knockout mice. Egr-1–/– BMMCs express similar levels of surface c-kit and IgE receptor as compared with those on Egr-1+/+ BMMCs. Importantly, IgE + TNP-induced TNF and IL-13 expression was significantly reduced at both mRNA and protein levels in Egr-1–/– BMMCs as compared with those in Egr-1+/+ BMMCs. Thus, our results suggest that de novo synthesis of Egr-1 represents a novel mechanism in FcϵRI signaling and is required for the full responsiveness of IgE-dependent TNF and IL-13 production by mast cells.

Carbon monoxide rescues ischemic lungs by interrupting MAPK-driven expression of early growth response 1 gene and its downstream target genes

Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signal-regulated kinase (ERK) activation, Egr-1 expression, and Erg DNA-binding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-alpha. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro, hypoxia-induced Egr-1 mRNA expression was ERK-dependent, and CO-mediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with l-nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP/protein kinase A- and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.

Hyperoxia upregulates Egr‐1 expression in cultured alveolar cells

Early growth response gene (Egr-1) is a stress response gene, activated by forms of stress and growth factor signaling. In mouse alveolar cells LA4, hyperoxia increased Egr-1 mRNA expression and protein content. Hyperoxia also activated EGR-Luc reporter gene. Blockade of MEK signaling prevented Egr-1 activation by hyperoxia. When cells were pretreated for 30 min with inhibitors of MEK/ERK pathway (PD 98059; 50 μM), p38 pathway (SB 203580; 10μM) and inhibitor of PI3K Wortmannin (100 nM), followed by 1 h hyperoxia and Northern blotting, only PD98059 prevented Egr-1 upregulation. Hyperoxia further phosphorylated ERK ½ kinase as demonstrated by Western blotting performed with phosphospecific antibodies against phosphor- ERK ½. Treatment of LA4 cells with EGFR specific inhibitors PD 153035 and 158780 blocked Egr1 mRNA upregulation. These results suggest that EGFR signaling mediates Egr-1 activation during hyperoxia. It remains to be established what are the downstream targets of Egr-1 and how does Egr-1 activation affect cellular adaptations and survival during hyperoxia.

Impact of Room Air Resuscitation on Early Growth Response Gene-1 in a Neonatal Piglet Model of Cerebral Hypoxic Ischemia

Early growth response gene-1 (Egr-1) is up-regulated by hypoxia-ischemia (HI) and reactive oxygen species (ROS) in adult animals, functioning as a master switch in inflammation and thrombogenesis. We hypothesized that resuscitation from HI with 100% O2 would result in greater Egr-1 expression, ROS, and cell death (CD) in the brains of newborn piglets than 21% O2. Two control groups breathed 21% O2 for 1 h followed by 21% or 100% O2 for 1 h. Two HI groups underwent carotid artery occlusion and breathed 8-12% O2 for 1 h followed by occlusion release and 21% or 100% O2 for 1 h. Brain Egr-1 mRNA and protein were analyzed via quantitative PCR and Western blot. CD and ROS were measured by fluorescence microscopy. Egr-1 mRNA expression increased throughout the brain in response to HI with regional heterogeneity, but protein levels did not. Resuscitation with 100% oxygen did not cause any additional Egr-1 mRNA, Egr-1 protein, CD, or ROS production as compared with 21% oxygen. There was no difference in physiologic recovery after HI with room air compared with 100% O2 resuscitation. However, 100% O2 administration was associated with increased CD in the brainstem independent of HI. Therefore, 100% O2 may have been toxic to some brainstem cells and potentially have significance in long-term neurologic sequelae seen after neonatal HI/resuscitation. Egr-1 protein levels may be tightly regulated in an attempt to diminish neurotoxicity or to enhance plasticity at this stage of development.

Suppression of early growth response factor-1 with egr-1 antisense oligodeoxynucleotide aggravates experimental duodenal ulcers

Previously, we demonstrated that cysteamine releases endothelin-1 in the rat duodenal mucosa, followed by increased expression of early growth response factor-1 (egr-1). We hypothesized that egr-1 is a key mediator gene in the multifactorial mechanisms of duodenal ulcer development and healing because its protein, transcription factor product Egr-1, regulates the expression of angiogenic growth factors. We wanted to determine the effect of egr-1 antisense oligonucleotide on cysteamine-induced duodenal ulcers as well as on the expression of bFGF, PDGF, and VEGF, of which synthesis is modulated by Egr-1. An antisense oligonucleotide to egr-1 was used to inhibit the synthesis of Egr-1 and to determine its effect on ulcer formation in the rat model of cysteamine-induced duodenal ulceration. Real-time RT-PCR and Western blot analysis were used to assess the expression of Egr-1 mRNA and protein as well as ERK, bFGF, PDGF, and VEGF. The antisense Egr-1 oligonucleotide inhibited the expression of egr-1 mRNA and protein and increased the duodenal ulcer size from 8.1 +/- 1.8 mm(2) in controls to 20.7 +/- 4.0 mm(2) (P < 0.01). Cysteamine induced phosphorylation of ERK1/2 and enhanced the synthesis of bFGF, PDGF, and VEGF in the preulcerogenic stages of duodenal ulceration, whereas egr-1 antisense oligonucleotide markedly decreased the expression of these growth factors in the duodenal mucosa. We also demonstrated that Egr-1 expression relates to the ulcerogenic effect of cysteamine because these actions were not exerted by the toxic analog ethanolamine. Thus Egr-1 seems to play a critical role in duodenal ulceration because Egr-1 downregulation aggravates experimental duodenal ulcers, most likely through the transcriptional inhibition of bFGF, PDGF, and VEGF synthesis.

Egr-1 Induces the Expression of Its Corepressor Nab2 by Activation of the Nab2 Promoter Thereby Establishing a Negative Feedback Loop

The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5'-region. The TATA- and initiator-less Nab2 promoter, located from -679 to -74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between -329 and -260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals.

Immediate-early gene expression in the central nucleus of the amygdala is not specific for anxiolytic or anxiogenic drugs

The lateral, basal, and central nuclei of the amygdala are part of a circuitry that instantiates many fear and anxious behaviors. One line of support indicates that immediate-early gene (IEG) expression (e.g., c-fos and egr-1 ( zif268)) is increased in these nuclei following fear conditioning. Other research finds that anxiogenic drugs working through various mechanisms induce IEG expression in the central nucleus of the amygdala (CeA) suggesting that expression is a neural marker for fear and anxiety. However, several studies have also found that anxiolytic drugs induce IEG expression in the CeA. Expression of egr-1 in the CeA and lateral nucleus of the amygdala following administration of anxiolytic and anxiogenic benzodiazepine and serotonin agonists and antagonists was investigated. The first experiment determined behaviorally active anxiolytic and anxiogenic doses for two anxiogenic drugs (FG 7142 and mCPP) and two anxiolytic drugs (diazepam and buspirone). The effects of anxiogenic and anxiolytic doses of these drugs on egr-1 expression in the amygdala were then tested in a second experiment. All four drugs increased egr-1 in the CeA indicating that increased egr-1 mRNA expression in the CeA is not specific to anxiolytic or anxiogenic effects of the drugs. We suggest that IEG expression in the CeA may be due to activation of circuits that are associated with systemic physiological homeostasis perturbed by a number of drugs including anxiogenic and anxiolytic compounds.

Cyclic GMP-dependent Protein Kinase Regulates CCAAT Enhancer-binding Protein β Functions through Inhibition of Glycogen Synthase Kinase-3

The CCAAT enhancer-binding protein (C/EBPbeta) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPbeta participates in cGMP-regulated transcription of c-fos in osteoblasts (Chen, Y., Zhuang, S., Cassenaer, S., Casteel, D. E., Gudi, T., Boss, G. R., and Pilz, R. B. (2003) Mol. Cell. Biol. 23, 4066-4082). In the present work, we show that cGMP/cGMP-dependent protein kinase (PKG) induced dephosphorylation and activation of C/EBPbeta by inhibiting glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta on Ser9 negatively regulates the enzyme activity, and we found that PKG phosphorylated this site both in vitro and in vivo; the in vivo phosphorylation occurred rapidly and preceded C/EBPbeta dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3beta is a C/EBPbeta kinase in resting cells. We determined that GSK-3beta phosphorylated C/EBPbeta in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPbeta was phosphorylated on these same sites in intact, unstimulated osteoblasts, and phosphorylation was decreased in cGMP-treated cells. Mutation of the GSK-3 phosphorylation sites in C/EBPbeta prevented C/EBPbeta phosphorylation in resting cells, enhanced C/EBPbeta DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPbeta. cGMP regulation of C/EBPbeta was disrupted by a mutant GSK-3beta(Ala9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPbeta by preventing the negative effects of GSK-3 phosphorylation.

Transcriptional Regulation of Activating Transcription Factor 3 Involves the Early Growth Response-1 Gene

Previously, our laboratory identified activating transcription factor 3 (ATF3) as up-regulated by nonsteroidal anti-inflammatory drugs using microarray analysis of mRNA from human colorectal cancer cells treated with sulindac sulfide. ATF3 is a transcription factor involved in cell growth, apoptosis, and invasion and is induced by a variety of anticancer and dietary compounds. However, the regulation of ATF3 by anticancer agents is not known. The promoter of ATF3 contains several transcription factor binding sites. We identified three putative Egr-1 binding sites in the promoter of ATF3 and report for the first time that the molecular mechanism responsible for the transcriptional regulation of ATF3 by two divergent pharmaceutical compounds, sulindac sulfide and troglitazone, involved the early growth response gene-1 (Egr-1). For example, overexpression of Egr-1 protein induced ATF3 mRNA 3.5-fold and transcriptional activity of an ATF3 promoter construct more than 20-fold. ATF3 and Egr-1 mRNA and protein and ATF3 promoter activity were induced by these compounds, whereas induction of ATF3 by these compounds was blocked by Egr-1 small interfering RNA. Sulindac sulfide and troglitazone regulated ATF3 promoter activity, which was suppressed when the two Egr-1 sites were mutated. These compounds induced phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2), whereas a dominant-negative inhibitor of mitogen-activate protein kinase kinase (MEK) 1 blocked the induction of ATF3. The MEK1/2 inhibitor PD98059 (2'-amino-3'-methoxyflavone) blocked the induction of ATF3 and Egr-1 mRNA expression and ATF3 promoter activity by these compounds. Therefore, this is a novel first report demonstrating that the expression of ATF3 occurs via Egr-1 downstream of Erk1/2.

PPAR-γ agonist rosiglitazone decreases focal cerebral ischemia-induced inflammation and brain damage in adult mice

Acute inflammation contributes to brain damage after focal cerebral ischemia. Although several transcription factors are known to be upregulated in the post-ischemic brain, the role of transcriptional events in controlling cerebral inflammation is not evaluated in detail. Peroxisome proliferation activated receptor- (PPAR-) is a ligand activated transcription factor of nuclear hormone receptor superfamily. When a ligand binds to PPAR-, it dimerizes with retinoic acid-X-receptor (RXR) to form a heterodimeric complex that binds to the cis-acting sequences (peroxisome proliferator response elements) on DNA to modulate the transcription of target genes. Thiozolinediones rosiglitazone and pioglitazone are potent exogenous agonists of PPAR-. Rosiglitazone and pioglitazone are currently FDA-approved for the treatment of type-2 diabetes. PPAR- agonists were shown to control inflammation associated with gut, myocardial and lung ischemia by blocking microglia/macrophage activation and pro-inflammatory gene expression. We evaluated the efficacy of rosiglitazone in preventing brain damage following transient middle cerebral artery occlusion (MCAO) in adult C57/BL6 mice. Real-time PCR analysis showed significant increases (by 2.7 to 4.2 fold; p<0.05; n of 6/group) in the PPAR- and RXR mRNA expression in the ipsilateral cortex compared to the contralateral cortex of mice at 6 h to 72 h of reperfusion following a 2 h transient MCAO. Compared to vehicle treated controls, 2 mg/Kg rosiglitazone pretreated mice (2 doses; at 4 h before MCAO and start of reperfusion) showed significantly smaller infarcts (by 57%9%, p<0.05; n of 10 mice/group). A 2 dose post-treatment (at 1 min and 2 h of reperfusion) also decreased the infarct volume by 52%8% (p<0.05; n of 9 mice/group) compared to vehicle control. Neuroprotection was also observed with a 1 dose rosiglitazone (2 mg/Kg) injected at either 1 min (by 46%8%, p<0.05; n of 10 mice/group) or 2 h of reperfusion (by 37%7%, p<0.05; n of 9 mice/group). In these mice, post-ischemic neurological deficits estimated on a 6-point scale were none to mild in the rosiglitazone groups compared to moderate to severe neurological deficits observed in the vehicle groups. Real-time PCR analysis showed a significant decrease in the post-ischemic mRNA expression of the pro-inflammatory transcription factors Egr-1 and c-EBP- and inflammatory genes IL-1, IL-6, MCP-1, ICAM-1 and iNOS in the ipsilateral cortex of rosiglitazone-treated mice compared to vehicle treated controls (n of 6/group). Furthermore, the ipsilateral cortex of the rosiglitazone treated mice (2 mg/Kg at 5 min reperfusion) showed significantly decreased numbers of activated macrophages/microglia (identified by ED-1 and CD11b immunostaining), compared to vehicle treated mice. These studies showed that PPAR- activation by its ligand is a potent anti-inflammatory mechanism that can decrease the post-ischemic brain damage. Rosiglitazone potassium salt used in these studies was purchased from the Cayman Chemicals USA.

Interleukin-10 Suppresses Tissue Factor Expression in Lipopolysaccharide-Stimulated Macrophages via Inhibition of Egr-1 and a Serum Response Element/MEK-ERK1/2 Pathway

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5'-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Expression changes of early response genes in lung due to high volume ventilation.

The expression changes of early response genes due to ventilation with high volume in adult rats in vivo were observed. Forty SD male rats were randomly divided into control and 30, 60, 90 and 120 min ventilation groups, respectively (n=8 in each group). The animals were ventilated with tidal volume of 42 ml/kg and a PEEP level of 0 cmH2O at a rate of 40 breaths per minute in room air with a ventilator was given to the small animals. The expression of Egr-1, C-jun and IL-1beta mRNA and proteins was detected by RT-PCR and immunohistochemical technique, respectively. The pathological changes in lung tissues were examined by HE staining. The results indicated that the expression of Egr-1, C-jun and IL-1beta mRNA was detectable at 30th min after overventilation, but there was no significant difference in comparison with that in control group until overventilation for 60 min. However, at 90 and 120 min there was a significent increase as compared with 30 min or control group (P<0.05). The expression of Egr-1, C-jun and IL-1beta deteced by immunohistochemical assay also showed a similar tendency of the gradual increase. In the 120 min ventilation group, the expression intensity of Egr-1, C-jun and IL-1beta proteins in lung cells was the strongest and the nuclear translocation was increased markedly in comparison with any other groups (P<0. 05). HE staining suggested that the degree of lung injury was aggravated gradually with the ventialtion going on and had a similar tendency to the expression of these early response genes and proteins. The current data suggested that overventilation activated and upregulated the expression of early response genes and the expression of these genes may be taken as the early signal to predict the onset and degree of lung injury. These results may demonstrated partially that the expression of early response genes induced by the mechanical stretch is associated with biochamic lung injury.

Expression of egr-1 ( zif268) mRNA in select fear-related brain regions following exposure to a predator

Research has demonstrated that immediate-early genes/inducible transcriptional factors (e.g., c-fos, egr-1) are increased in amygdala nuclei (lateral, basal and central nuclei) known to be involved in fear conditioning, footshock stress and novelty. Although these data suggest that expression of inducible transcriptional factors are involved in fear, other non-shock ethologically based paradigms (predator or predator odor exposure) do not appear to increase c-fos in the lateral and basal nuclei. While the lack of c-fos expression may indicate that predator stress does not engage the lateral and basal amygdala nuclei, it may be that c-fos in the amygdala is not responsive to predator exposure. Therefore, egr-1, which increases in the lateral nucleus following fear conditioning, footshock and novelty, was assessed to determine if its expression is induced in rats exposed to a cat. Five minutes of cat exposure did not increase expression of egr-1 mRNA in the lateral nucleus of the amygdala. egr-1 was increased in the paraventricular nucleus of the hypothalamus, indicating cat-induced stress, and visual cortex compared to rats that were either confined for 5 min or handled. In the lateral periaqueductal gray, handled rats displayed a left hemisphere dominance, which disappeared in both the cat-exposed and confined group, suggesting that immobility, induced by either cat-induced stress or unstressed confinement, increased right hemisphere egr-1 expression. The results are discussed in a context of differences and similarities in neural circuitry for conditioned and unconditioned fear.

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

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

Chylomicron remnants regulate early growth response factor-1 in vascular smooth muscle cells

Early growth response factor-1 (Egr-1) is a zinc-finger transcription factor that induces genes that promote atherosclerosis. The goal of the present study was to determine whether Egr-1 expression is modulated by atherogenic, triglyceride rich lipoproteins known as chylomicron remnants. Chylomicron remnants induced Egr-1 mRNA and protein expression in rat cultured vascular smooth muscle cells (VSMCs) and activated extracellular signal-regulated kinase (ERK) 1/2 in VSMCs. Further, chylomicron remnant-induced Egr-1 expression was inhibited by PD98059, a selective inhibitor of MAPK kinase (MEK), suggesting that the action of chylomicron remnants on Egr-1 was dependent on the ERK/MEK pathway. Chylomicron remnants also induced mRNA expression of the pro-inflammatory cytokines, IL-2 and IFN-γ in VSMCs. We conclude that chylomicron remnants act as atherogenic lipoproteins via induction of Egr-1 expression and via cytokine-mediated inflammation.

Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance

To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance. A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison. After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF. Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF.

In Vivo and In Vitro Silica Induces Nuclear Factor Egr-1 Activation Mediated by ERK 1/2 in RAW264.7 Cell Line

The transcription factor early growth response gene (Egr-1) is a stress response gene activated by various forms of stress. The effect of silica on transcription and expression of Egr-1 was investigated in rat lung and in RAW264.7 cells. Silica induced the expression of Egr-1 in vivo and was mainly located in alveolar macrophage cells and lung epithelial cells. Furthermore, silica induced Egr-1 mRNA and protein expression in cultured RAW264.7 cells. Immunofluorescence microscopy revealed translocation of Egr-1 to the nucleus in response to silica. The contribution of the extracellular signal-regulated kinase (ERK) pathway to the activation of Egr-1 in response to silica was examined. Exposure to silica resulted in a rapid phosphorylation of ERK 1/2 kinases in RAW264.7 cells. MAP Kinase Kinase (MEK) inhibitor U0126 prevented Egr-1 induction by silica. The results suggest that silica could induce Egr-1 activation in macrophages in vivo and in vitro and that phosphorylated ERK 1/2 may be involved in this action.