Egr-1 Response Research Articles

Androgen receptor and hyaluronan-mediated motility receptor as new molecular targets of baicalein: new molecular mechanisms for its anticancer properties.

Natural compounds known as phytochemicals have served as valuable resources for the development of new anti-cancer drugs and treatment of malignancies. Among these phytochemicals, baicalein is an emerging anti-tumor flavonoid obtained from Scutellaria baicaleinsis (Lamiaceae), but its underlying mechanisms of action and molecular targets have not yet been completely elucidated. Here, we identified new mechanisms for the anti-tumor activities of baicalein, providing evidence that hyaluronan-mediated motility receptor (HMMR) and androgen receptor (AR) are new molecular targets of baicalein in human cancer cells. We observed that HMMR, known to be highly associated with poor prognosis in a wide range of human cancers, was substantially downregulated by baicalein at mRNA and protein levels. Reporter assays further revealed that the suppression of HMMR by baicalein might occur through a transcriptional regulatory mechanism with the participation of Egr-1, E2F3α, and serum response factor (SRF). We also found that baicalein significantly inhibits androgenic responses in hormone-responsive prostate cancer cells, indicating that this might be attributed to the downregulation of AR promoter activity by baicalein. Additionally, baicalein markedly induced the expression of tumor suppressive miR-30C, which might be partly involved in baicalein-mediated autophagy and anti-cancer effects. Overall, our study sheds light on new diverse mechanisms of the anti-cancer effects exhibited by baicalein, implying that baicalein could be a potential therapeutic agent against human cancers and function as an inhibitor of HMMR and AR.

Functional Integration of Adult-Generated Neurons in Diabetic Goto-Kakizaki Rats.

Adult-born neurons in the dentate gyrus (DG) make important contributions to learning as they integrate into neuronal networks. Neurogenesis is dramatically reduced by a number of conditions associated with cognitive impairment, including type 2 diabetes mellitus (T2DM). Increasing neurogenesis may thus provide a therapeutic target for ameliorating diabetes-associated cognitive impairments, but only if new neurons remain capable of normal function. To address the capacity for adult-generated neurons to incorporate into functional circuits in the hyperglycemic DG, we measured Egr1 expression in granule cells (GCs), BrdU labeled four weeks prior, in Goto-Kakizaki (GK) rats, an established model of T2DM, and age-matched Wistars. The results indicate that while fewer GCs are generated in the DG of GK rats, GCs that survive readily express Egr1 in response to spatial information. These data demonstrate that adult-generated GCs in the hyperglycemic DG remain functionally competent and support neurogenesis as a viable therapeutic target.

Sustained Egr-1 Response via p38 MAP Kinase Signaling Modulates Early Immune Responses of Dendritic Cells Parasitized by Toxoplasma gondii.

As a response to a diverse array of external stimuli, early growth response protein 1 (Egr-1) plays important roles in the transcriptional regulation of inflammation and the cellular immune response. However, a number of intracellular pathogens colonize immune cells and the implication of Egr-1 in the host-pathogen interplay has remained elusive. Here, we have characterized the Egr-1 responses of primary murine and human dendritic cells (DCs) upon challenge with the obligate intracellular parasite Toxoplasma gondii. We report that live intracellular parasites induce a sustained high expression of Egr-1 in DCs, different from the immediate-early Egr-1 response to parasite lysates, inactivated parasites or LPS. Moreover, a distinct nuclear localization of elevated amounts of Egr-1 protein was detected in infected DCs, but not in by-stander DCs. The ERK1/2 MAPK signaling pathway mediated the canonical immediate-early Egr-1 response to soluble antigens in a MyD88/TLR-dependent fashion. In contrast, a non-canonical extended Egr-1 response that relied primarily on p38 MAPK signaling was induced by intracellular parasites and was exhibited similarly by MyD88-deficient and wildtype DCs. The extended phase Egr-1 response was dramatically reduced upon challenge of DCs with T. gondii parasites deficient in GRA24, a secreted p38-interacting protein. Further, Egr-1-silenced primary DCs maintained their migratory responses upon T. gondii challenge. Importantly, Egr-1 silencing led to elevated expression of co-stimulatory molecules (CD40, CD80) in Toxoplasma-infected DCs and in LPS-challenged immature DCs, indicating that Egr-1 responses suppressed maturation of DCs. Moreover, the IL-12 and IL-2 responses of Toxoplasma-challenged DCs were modulated in a GRA24-dependent fashion. Jointly, the data show that the Egr-1 responses of DCs to microbial external stimuli and intracellular stimuli can be selectively mediated by ERK1/2 or p38 MAPK signaling, and that Egr-1 can act as an intrinsic negative modulator of maturation in primary DCs.

Genetic Targeting and Chemogenetic Inhibition of Newborn Neurons.

The aim of this study was to develop a method to silence a very specific set of cells in a spatially and temporally refined manner. Here, an approach is presented that combines the use of a transgenic mouse line, expressing cre recombinase under a nestin promoter, with lentiviral delivery of a floxed, ivermectin (IVM)-gated chloride channel construct to the dentate gyrus. This approach was used to express an IVM-sensitive chloride channel in newly born granule cells in adult mouse brains, and its ability to silence neuronal activity was tested by analyzing the effect on immediate early gene expression in vitro in cre-transgenic primary neuronal cultures. IVM treatment of cells expressing the chloride channel prevented gabazine-induced expression of the immediate early gene product EGR1, while cells expressing a control inactive channel or no channel retained their EGR1 response. Thus, a genetic strategy is presented for targeting a specific neurogenic niche for transgene expression in the adult mouse brain, and proof of principle is shown that it can be used in vitro as a method for silencing neuronal activity.

Egr-1 regulates RTA transcription through a cooperative involvement of transcriptional regulators.

Kaposi’s sarcoma associated herpesvirus (KSHV) regulates the host cellular environment to establish life-long persistent infection by manipulating cellular signaling pathways, with approximately 1- 5% of cells undergoing lytic reactivation during the course of infection. Egr-1 (Early Growth Response Factor-1) is one such cellular transcription factor, which gets phosphorylated during the lytic phase of viral life cycle to perpetrate its function. This study demonstrates the mechanism of how Egr-1 mediates transcription of the immediate early gene, RTA (Replication and transcription activator), which is the lytic switch gene of KSHV. Egr-1 depleted KSHV infected cells exhibited reduced expression of RTA. Also, an increase in Egr-1 phosphorylation led to a higher virion production, which was suppressed in the presence of p38 and Raf inhibitors. Reporter assays showed that coexpression of Egr-1 and CBP (CREB-binding protein) enhances RTA promoter activity as compared to the expression of either Egr-1 or CBP alone. Binding of Egr-1 and CBP at RTA promoter was analyzed by chromatin immunoprecipitation assay (ChIP), which showed an enhanced accumulation during viral reactivation. Mutation in Egr-1 binding site of the RTA promoter eliminated Egr-1 response on promoter activation. Furthermore, de novo infection of THP-1 (monocytic) and HUVECs (endothelial) cells showed an upregulation of Egr-1 phosphorylation, whereas depletion of Egr-1 reduced the mRNA levels of RTA during primary infection. Together, these results demonstrate a cooperative role of Egr-1 and CBP in mediating RTA transcription, which significantly improves our understanding of the involvement of cellular factors controlling RTA transcription in KSHV pathogenesis.

Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide

Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.

Stress in utero alters neonatal stress-induced regulation of the synaptic plasticity proteins Arc and Egr1 in a sex-specific manner.

The present study in juvenile rats investigated a "two-hit model" to test the impact of prenatal stress exposure ("first hit") on the regulation of the synaptic plasticity immediate early genes Arc and Egr1 in response to a second neonatal stressor ("second hit") in a sex-specific manner. Three stress-exposed animal groups were compared at the age of 21days in relation to unstressed controls (CON): preS animals were exposed to various unpredictable stressors during the last gestational trimester; postS animals were exposed to 45min restraint stress at postnatal day 21, pre/postS animals were exposed to a combination of pre- and postnatal stress as described for the two previous groups. The postS and pre/postS groups were killed 2h after exposure to the postnatal stressor, males and females were separately analyzed. In line with our hypothesis we detected sex-specific stress sensitivity for both analyzed proteins. Males did not show any significant changes in Arc expression irrespective of the stress condition. In contrast, females, which had been pre-exposed to prenatal stress, displayed an "amplified" Arc upregulation in response to postnatal stress (pre/postS group) compared to unstressed controls, which may reflect a "sensitization" effect of prenatal stress. For Egr1, the females did not show any stress-induced regulation irrespective of the stress condition, whereas in males, which were pre-exposed to prenatal stress, we observed a "protective" effect of prenatal stress on postnatal stress-induced downregulation of Egr1 expression (pre/postS group), which may indicate that prenatal stress exposure may induce "resilience".

Genome‐wide analysis of gene transcription by EGR1 in response to TGF‐β2 stimulation of small intestinal cell differentiation (623.23)

Human milk contains growth factors that maintain intestinal mucosal homeostasis. However, the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. We used IEC‐6 (intestinal crypt cell) cells as a model and treated them with 10 ng/ml TGF‐β2, a milk growth factor known for its stimulatory effect on intestinal cell differentiation. Immunoblotting and immunofluorescent assays showed that EGR‐1 (early immediate gene, regulating cell survival, growth, differentiation and transformation) expression in the nucleus was stimulated, with maximum expression occurring at 1h and then a decrease with time. EGR1 was predicted to have binding sites in the promoters of Axin1 (‐248bp/258bp, the scaffold protein in Wnt signaling), LRP5 (‐78bp/113bp, interacting with Axin1), Notch1 (‐76bp/84bp, Notch signaling) and Hes1 (‐1158bp/1170bp, affecting differentiation of intestinal cells). Chromatin immunoprecipitation (CHIP) verified a binding site in the Axin1 promoter. To comprehensively explore the functions of EGR1 in intestinal cell growth, next‐generation sequencing (NGS) was used to examine the interaction between EGR1 and other genes under the stimulation of TGF‐β2 at 0h, 1h and 48h, respectively. In conclusion, our results for the first time show the genome‐wide interaction between EGR1, a TGF‐ β2 responsive factor, and active genes involved in intestinal crypt cell growth and differentiation.

Egr-1 induction provides a genetic response to food aversion in zebrafish

As soon as zebrafish larvae start eating, they exhibit a marked aversion for bitter and acidic substances, as revealed by a consumption assay, in which fluorescent Tetrahymena serve as a feeding basis, to which various stimuli can be added. Bitter and acidic substances elicited an increase in mRNA accumulation of the immediate-early response gene egr-1, as revealed by in situ hybridization. Conversely, chemostimulants that did not induce aversion did not induce egr-1 response. Maximum labeling was observed in cells located in the oropharyngeal cavity and on the gill rakers. Gustatory areas of the brain were also labeled. Interestingly, when bitter tastants were repeatedly associated with food reward, zebrafish juveniles learned to ingest food in the presence of the bitter compound. After habituation, the acquisition of acceptance for bitterness was accompanied by a loss of egr-1 labeling. Altogether, our data indicate that egr-1 participates specifically in food aversion. The existence of reward-coupled changes in taste sensitivity in humans suggests that our results are relevant to situations in humans.

Vision-guided ocular growth in a mutant chicken model with diminished visual acuity

Visual experience is known to guide ocular growth. We tested the hypothesis that vision-guided ocular growth is disrupted in a model system with diminished visual acuity. We examine whether ocular elongation is influenced by form-deprivation (FD) and lens-imposed defocus in the Retinopathy, Globe Enlarged (RGE) chicken. Young RGE chicks have poor visual acuity, without significant retinal pathology, resulting from a mutation in guanine nucleotide-binding protein β3 (GNB3), also known as transducin β3 or Gβ3. The mutation in GNB3 destabilizes the protein and causes a loss of Gβ3 from photoreceptors and ON-bipolar cells (Ritchey et al., 2010). FD increased ocular elongation in RGE eyes in a manner similar to that seen in wild-type (WT) eyes. By comparison, the excessive ocular elongation that results from hyperopic defocus was increased, whereas myopic defocus failed to significantly decrease ocular elongation in RGE eyes. Brief daily periods of unrestricted vision interrupting FD prevented ocular elongation in RGE chicks in a manner similar to that seen in WT chicks. Glucagonergic amacrine cells differentially expressed the immediate early gene Egr1 in response to growth-guiding stimuli in RGE retinas, but the defocus-dependent up-regulation of Egr1 was lesser in RGE retinas compared to that of WT retinas. We conclude that high visual acuity, and the retinal signaling mediated by Gβ3, is not required for emmetropization and the excessive ocular elongation caused by FD and hyperopic defocus. However, the loss of acuity and Gβ3 from RGE retinas causes enhanced responses to hyperopic defocus and diminished responses to myopic defocus.

P53-Independent Anti-Myeloma Activity of Prima-1met

Abstract 1826 Background:Although mutation/deletion of p53 is relatively rare (∼10%) in multiple myeloma (MM), this subset of patients are resistant to current therapies with very poor prognosis. Thus, improved therapeutic strategy is urgently required for this high risk group of patients. Prima-1met (p53 reactivation and induction of massive apoptosis), a methylated derivative and more active analog of prima-1, is a small molecule which restores tumor suppressor function to mutant p53 and has shown to induce cytotoxic effects and apoptosis in various human tumor cells harboring mutant p53. However, anti-myeloma activity of prima-1met is unknown. Experimental design:6 human MM cell lines, primary MM samples from 7 newly diagnosed patients, bone marrow mononuclear cells (BMNCs) and peripheral blood mononuclear cells (PBMNCs) from 3 healthy donors were used for this study. MM.1S and H929 cell lines harbour wild type p53; 8266, U266, and LP1 express mutant p53; and 8226R5 does not express p53 (p53 null). The cells were treated with different dose (2.5-100 μM) of prima-1met for different time periods. The therapeutic effect of prima-1met was studied in these cells using MTT cell viability assay, flow cytometry (FCM), qRT-PCR, and Western blot (WB) analysis. Results:Prima-1met efficiently inhibited the viability of MM cell lines irrespective of p53 status. Similarly, prima-1met induced a dose-dependent cytotoxic response in 5 of the 7 patient samples tested. However, prima-1met only showed limited cytotoxicity toward normal BMNCs or PBMNCs suggesting a preferential killing of MM cells by prima-1met. We next examined whether the declining in the viability of the cells was due to the apoptosis induction by prima-1met. Depending on the cell types, 48 hrs after treatment with 100 μM prima-1met 35–70% Annexin V-positive cells was determined by FCM. The cytotoxic effect of prima-1met was associated with activation of caspase-3, PARP cleavage, up-regulation of Noxa and c-Jun without significant modulation of p53 level. Treatment of cells with prima-1met induced the activation of caspase-8 but not -9 suggesting the association of extrinsic pathway. Furthermore, the pan-caspase inhibitor, Z-VAD-FMK, inhibited the activation of caspase-3 indicating that prima-1met-induced apoptosis in MM cells is caspase-dependent. The apoptotic effect of prima-1met was not significantly affected in H929 cells transfected with p53 siRNA confirming that prima-1met exerts anti-myeloma activity in a p53-independent manner. To further explore the molecular mechanisms associated with prima-1met-induced apoptosis, RNA from DMSO-treated and prima-1met-treated MM cells (MM.1S, U266 and 8266R5) was profiled by Illumina HT-12 microarray and differentially expressed genes were analysed. A significant number of genes associated with stress response such as HASP1B, HSPA6, FOS, ATF3, MYC and EGR-1 were modulated in all 3 cell lines upon prima-1met treatment. Of note, EGR-1 is highly up-regulated (7 to 21-fold) in all 3 cell lines. EGR-1 is a member of the immediate-early gene family and plays a pivotal role in the regulation of cell growth, differentiation and apoptosis. Overexpression of EGR-1 has shown to down-regulate anti-apoptotic protein survivin and induction of apoptosis in myeloma cells. The role of EGR-1 and other relevant stress response genes associated with p53-independent apoptosis induced by prima-1met is currently under investigation.In addition, we found a strong synergistic effect in MM cells harbouring either wild type or mutant p53 when prima-1met was combined with dexamethasone (DXM) or doxorubicin (DOXO). We examined cell cytotoxicity of the combination by using prima-1met and DOXO/DXM at concentrations lower than their maximal cytotoxic concentrations. The combination of 2 μM prima-1met and 0.5 μM DOXO/DXM produced a synergistic response (CI=0.82-0.87) in H929 cells. The combination of 10 μM prima-1met and 2.0 μM DOXO displayed a synergistic cytotoxic response (CI=0.68) in U266 cells. Conclusion:This study is the first to show the potential p53-indpendent anti-myeloma activity of prima-1met. Our results provide the framework for the clinical evaluation of prima-1met either alone or in combination with other chemotherapeutic agents which may offer a novel and more efficient therapeutic strategy for the treatment of MM patients carrying either wild type or mutant p53. Disclosures:No relevant conflicts of interest to declare.

Neurotensin induced Egr-1 activity is altered in the postpartum period in mice

Neurotensin (NT) is a 13 amino acid neuropeptide that is identical in mice and humans and is released from and acts upon a number of social brain regions. Recent work indicates NT neurotransmission may be altered in postpartum females and support the onset of some maternal behaviors. In a recent study, we highlighted how virgin and postpartum brains from mice selected for high offspring protection differ in response to injected NT (0.1 μg) relative to vehicle when examining c-Fos profiles across the CNS. In this companion study we use a second marker for brain activity, Egr-1, and evaluate multiple brain regions. Common significant increased Egr-1 responses to NT (relative to vehicle) were found in both female groups only in ventromedial hypothalamus. In lateral periaqueductal gray, virgin mice showed a significant Egr-1 increase with NT (relative to vehicle), but maternal mice did not. When comparing NT injections, virgin (relative to maternal) mice had significantly higher activity in five regions, including anterior hypothalamus, lateral hypothalamus, somatosensory cortex, paraventricular nucleus, and zona incerta; no regions were higher in maternal mice. A Principal Components Analysis was also used for data mining and in virgin mice, greater changes in activity hubs were found with NT (relative to vehicle) than for maternal mice. Overall, a lower sensitivity to NT in terms of Egr-1 reactivity in the maternal state was highlighted and this is consistent with previous c-Fos results. These findings provide additional insight into the mechanisms by which NT functions in the CNS.

Transcriptional Regulation of EGR1 by EGF and the ERK Signaling Pathway in Prostate Cancer Cells

The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes. Unlike other tumor types, prostate tumors express high levels of EGR1 relative to normal tissues. However, the mechanism of EGR1 regulation in prostate tumor cells is unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using RT-PCR to quantify EGR1 transcripts, we found that EGF induced EGR1 expression in a dose- and time-dependent manner and the ERK pathway inhibitor, PD98059, abrogated the EGF-mediated EGR1 response in LNCaP and PC3 cells. Analysis of the EGR1 promoter using deletion constructs identified an EGF-responsive region in the proximal promoter (-771 to -245 bp) containing 3 potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that Elk-1 binding at the SRE sites of the EGR1 promoter was enhanced by EGF treatment in PC3 cells. Overexpression of Elk-1 was sufficient to activate the EGF-responsive region of EGR1 promoter in PC3 cells and, similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity. Taken together, these results demonstrate for the first time that EGR1 expression in PC3 cells is mediated through an EGF-ERK-Elk-1 signaling cascade.

Ltor PROCESSES MALE TÚNGARA MATING CALLS

It's a warm Panamanian evening and male Physalaemus frogs are out crooning to their females. But with many other species serenading in the night's air, how do túngara females pick out their mates from the rest of the hullabaloo? Lisa Mangiamele and Sabrina Burmeister from the University of North Carolina at Chapel Hill explain that, `Sensory systems respond selectively to information in the environment – extracting some stimulus features while discarding others.' According to Burmeister, females respond to the drop in frequency as the male's whine glissandos from 900 to 400 Hz. They also respond equally well to a synthesised two-tone call that drops from 800 Hz to 500 Hz tone; the underlying two-tone signature is all that a túngara female needs to correctly select a mate of her own species. Knowing that a transcription factor, egr-1, is produced in brain tissue in response to stimulation, the duo decided to identify the regions of the female brain that are involved in processing the males' serenades by looking for evidence that the egr-1 gene had been activated in the brain after listening to natural and simulated mating calls (p. 2911).The duo found that one tiny region of the female's brain, the laminar nucleus of the torus semicircularis (Ltor), responded to both the males' whining glissando and the signature two-tone simulation, suggesting that Ltor is the `neural analyser for call recognition'. Other regions of the auditory brainstem and thalamus also responded to the males' full-spectrum call, but they failed to activate egr-1 in response to the two-tone signature, leading the duo to conclude, `Ltor activation is sufficient to explain species recognition decisions for female túngara frogs.'

Nemo through the looking-glass: a commentary on Desjardins & Fernald

Mirror-elicited aggression has been widely used in animal behaviour research. Recently, Desjardins & Fernald [[1][1]] reported that males of the cichlid fish Astatotilapia burtoni fighting a mirror had higher expression of immediate early genes in brain areas homologous to the amygdala and the

Abstract 4013: Transcriptional regulation of EGR1 by EGF and the ERK signaling pathway in prostate cancer

Abstract The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes resulting in diverse responses such as cell growth, proliferation, and apoptosis. Unlike other tumor types, in prostate tumor tissues EGR1 expression is elevated relative to normal tissues. However, the mechanism(s) of regulation of EGR1 in prostate tumor cells are unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using real-time PCR to quantify EGR1 transcripts, EGF induced EGR1 expression levels in a dose and time-dependent manner; and kinase inhibitors, PD98059 and wortmannin, abrogated the EGF-mediated EGR1 response. Analysis of the EGR1 promoter using reporter constructs identified an EGF-responsive region in the proximal promoter containing three potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that EGF enhanced Elk-1 binding at the SRE sites of the EGR1 promoter in PC3 cells, and Elk-1 also bound in the absence of stimulus. Overexpression of Elk-1 enhanced activation of the EGF-responsive region indicating that Elk-1 is sufficient to activate EGR1 in PC3 cells. Similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity indicating that Elk-1 is necessary for EGF-induced EGR1 transcription in both PC3 and LNCaP cells. Taken together, these results demonstrate that EGR1 expression in prostate cancer cells is mediated through an EGF-ERK-Elk-1 signaling cascade. Since elevated EGF and EGF receptor are associated with prostate cancer progression, these results may explain elevated EGR1 in high-grade prostate tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2011-4013

Restoring Cell Responsiveness to GM-CSF and IL-3 by PTEN Is Via Controlling the Regulation of Egr-1 In TF-1a Cell Line

AbstractAbstract 2504Cytokine independence is a common event in leukemia. The mechanism is often linked to deregulation of Ras/PI3K, Ras/MAP, and JAK/STAT pathways. PTEN, a major negative regulator of the Ras pathway, was initially isolated as a tumor suppressor in a variety of malignancies. We recently reported that PTEN is deficient in 67% of juvenile myelomonocytic leukemia (JMML) patients. Other groups have also found alterations of PTEN, PI3K, or AKT in 47.7% of T-cell acute lymphoblastic leukemia. Mice who are rendered pten-deficient develop myeloproliferative disorders and acute myeloid and lymphoid leukemias, and the leukemia-initiating stem cell in these mouse models could be distinguished from the normal hematopoietic stem cell by a differential response to rapamycin. The transcription factor, Egr-1, has been known to be a positive regulator of PTEN expression. It plays an important role in controlling proliferation as well as mobilization of hematopoietic stem cells. It is unknown whether PTEN had a feedback loop on regulating Egr-1 expression. Since poorly differentiated progenitors are often mobilized to the peripheral blood in leukemia patients, we hypothesized that PTEN deficiency alters the feedback loop of Egr-1 in response to cytokine stimulation. In this manner, PTEN deficiency might contribute to the cytokine independence observed in many types of leukemia. In order to evaluate this hypothesis we investigated the role of PTEN in cells responding to GM-CSF stimulation in the leukemia cell line, TF-1a. TF-1a is PTEN deficient as well as cytokine independent, and displays a constitutively high expression of Egr-1. We restored PTEN expression in TF-1a cells. This was accomplished by packaging the coding sequence of wild-type PTEN in a retrovirus vector, pBMN-GFP, that carried a gene encoding green fluorescent protein (GFP). TF-1a cells were transfected with pBMN-PTEN-GFP or pBMN-GFP by spinoculation. The pure population of transfected cells was sorted by FACS, followed by limiting dilution. The transfected single colonies were expanded for further evaluation of PTEN expression and biological function. Data from these experiments showed: 1) over-expression PTEN in TF-1a down-regulated the expression of Egr-1 and restored the responsiveness of Egr-1 to GM-CSF stimulation, 2) PTEN restored the responsiveness of TF-1a to GM-CSF and IL-3, and 3) these events are CREB-independent. In conclusion, our data demonstrates that PTEN contributes to the cell responsiveness to GM-CSF and IL-3, and has a negative feedback role to regulate Egr-1 expression in TF-1a cells. In addition to Egr-1 regulating PTEN in a positive manner, our data indicates the presence of a feedback loop from PTEN back to Egr-1. This appears to be an event independent of CREB. Disclosures:No relevant conflicts of interest to declare.

Neural Activity Patterns in Response to Interspecific and Intraspecific Variation in Mating Calls in the Túngara Frog

BackgroundDuring mate choice, individuals must classify potential mates according to species identity and relative attractiveness. In many species, females do so by evaluating variation in the signals produced by males. Male túngara frogs (Physalaemus pustulosus) can produce single note calls (whines) and multi-note calls (whine-chucks). While the whine alone is sufficient for species recognition, females greatly prefer the whine-chuck when given a choice.Methodology/Principal FindingsTo better understand how the brain responds to variation in male mating signals, we mapped neural activity patterns evoked by interspecific and intraspecific variation in mating calls in túngara frogs by measuring expression of egr-1. We predicted that egr-1 responses to conspecific calls would identify brain regions that are potentially important for species recognition and that at least some of those brain regions would vary in their egr-1 responses to mating calls that vary in attractiveness. We measured egr-1 in the auditory brainstem and its forebrain targets and found that conspecific whine-chucks elicited greater egr-1 expression than heterospecific whines in all but three regions. We found no evidence that preferred whine-chuck calls elicited greater egr-1 expression than conspecific whines in any of eleven brain regions examined, in contrast to predictions that mating preferences in túngara frogs emerge from greater responses in the auditory system.ConclusionsAlthough selectivity for species-specific signals is apparent throughout the túngara frog brain, further studies are necessary to elucidate how neural activity patterns vary with the attractiveness of conspecific mating calls.

ERKs and JNKs Mediate Hydrogen Peroxide-Induced Egr-1 Expression and Nuclear Accumulation in H9c2 Cells

One of the most significant insults that jeopardize cardiomyocyte homeostasis is a surge of reactive oxygen species (ROS) in the failing myocardium. Early growth response factor-1 (Egr-1) has been found to act as a transcriptional regulator in multiple biological processes known to exert deleterious effects on cardiomyocytes. We thus investigated the signaling pathways involved in its regulation by H2O2. Egr-1 mRNA levels were found to be maximally induced after 2 h in H2O2-treated H9c2 cells. Egr-1 respective response at the protein level, was found to be maximally induced after 2 h of treatment with 200 microM H2O2, remaining elevated for 6 h, and declining thereafter. H2O2-induced upregulation of Egr-1 mRNA and protein levels was ablated in the presence of agents inhibiting ERKs pathway (PD98059) and JNKs (SP600125, AS601245). Immunofluorescent experiments revealed H2O2-induced Egr-1 nuclear sequestration to be also ERK- and JNK-dependent. Overall, our results show for the first time the fundamental role of ERKs and JNKs in regulating Egr-1 response to H2O2 treatment in cardiac cells at multiple levels: mRNA, protein and subcellular distribution. Nevertheless, further studies are required to elucidate the specific physiological role of Egr-1 regarding the modulation of gene expression and determination of cell fate.

CREB Deficiency: A Potential Critical Factor for Selective GM-CSF Hypersensitivity in Juvenile Myelomonocytic Leukemia.

Abstract 2604Poster Board II-580Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures:No relevant conflicts of interest to declare.