ETS-domain Transcription Factor Research Articles

Opposing actions of histone deacetylase 1 and Notch signalling restrict expression of erm and fgf20a to hindbrain rhombomere centres during zebrafish neurogenesis

The rate and pattern of neurogenesis in the developing vertebrate nervous system is controlled by a complex interplay of intercellular signalling pathways and transcriptional control mechanisms. In the zebrafish hindbrain, Fgf20a promotes transcription of the gene encoding the ETS-domain transcription factor Erm in the non-neurogenic centres of rhombomeres. Here, we demonstrate that the epigenetic regulator, Histone Deacetylase 1 (Hdac1) and the Notch signalling pathway have opposing functions in regulating expression of both erm and fgf20a in the zebrafish hindbrain. Our results show that Hdac1 is required for expression of erm and fgf20a in rhombomeres, and that the Hdac1-dependent expression of these two genes is attenuated in rhombomere boundary regions by Notch signalling activity, thereby restricting erm and fgf20a transcripts to narrow stripes of cells at rhombomere centres.

The ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma

BackgroundMany members of the ETS-domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-domain transcription factors. The PEA3 subfamily of ETS-domain transcription factors have been implicated in tumour metastasis in several different cancers.ResultsHere, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas.ConclusionsThis study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.

Differential Regulation of Dopaminergic Gene Expression byEr81

A recent study proposed that differentiation of dopaminergic neurons requires a conserved "dopamine motif" (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1

Fatty acid desaturases (FADS) play an important role in the formation of omega-6 and omega-3 highly unsaturated fatty acids (HUFAs). The composition of HUFAs in the human metabolome is important for membrane fluidity and for the modulation of essential physiological functions such as inflammation processes and brain development. Several recent studies reported significant associations of single nucleotide polymorphisms (SNPs) in the human FADS gene cluster with HUFA levels and composition. The presence of the minor allele correlated with a decrease of desaturase reaction products and an accumulation of substrates. We performed functional studies with two of the associated polymorphisms (rs3834458 and rs968567) and showed an influence of polymorphism rs968567 on FADS2 promoter activity by luciferase reporter gene assays. Electrophoretic mobility shift assays proved allele-dependent DNA-binding ability of at least two protein complexes to the region containing SNP rs968567. One of the proteins binding to this region in an allele-specific manner was shown to be the transcription factor ELK1 (a member of ETS domain transcription factor family). These results indicate that rs968567 influences FADS2 transcription and offer first insights into the modulation of complex regulation mechanisms of FADS2 gene transcription by SNPs.

Overlapping promoter targeting by Elk-1 and other divergent ETS-domain transcription factor family members.

ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.

Mouse plasmacytoma-expressed transcript 1 knock out induced 5-HT disruption results in a lack of cognitive deficits and an anxiety phenotype complicated by hypoactivity and defensiveness

Serotonin (5-HT) is involved in many developmental processes and influences behaviors including anxiety, aggression, and cognition. Disruption of the serotonergic system has been implicated in human disorders including autism, depression, schizophrenia, and ADHD. Although pharmacological, neurotoxin, and dietary manipulation of 5-HT and tryptophan hydroxylase has added to our understanding of the serotonergic system, the results are complicated by multiple factors. A newly identified ETS domain transcription factor, Pet-1, has direct control of major aspects of 5-HT neuronal development. Pet-1 is the only known factor that is restricted in the brain to 5-HT neurons during development and adulthood and exerts dominant control over 5-HT neuronal phenotype. Disruption of Pet-1 produces an ∼80% loss of 5-HT neurons and content and results in increased aggression in male Pet-1−/− mice [Hendricks TJ, Fyodorov DV, Wegman LJ, Lelutiu NB, Pehek EA, Yamamoto B, Silver J, Weeber EJ, Sweatt JD, Deneris ES (2003) Neuron 37:233–247]. We hypothesized that Pet-1−/− mice would also exhibit changes in anxiety and cognition. Pet-1−/− mice were hypoactive which may have affected the observed lack of anxious behavior in the elevated zero maze and light-dark test. Pet-1−/− mice, however, were more defensive during marble burying and showed acoustic startle hyper-reactivity. No deficits in spatial, egocentric, or novel object recognition learning were found in Pet-1−/− mice. These findings were unexpected given that 5-HT depleting drugs given to adult or developing animals result in learning deficits [Mazer C, Muneyyirci J, Taheny K, Raio N, Borella A, Whitaker-Azmitia P (1997) Brain Res 760:68–73; Morford LL, Inman-Wood SL, Gudelsky GA, Williams MT, Vorhees CV (2002) Eur J Neurosci 16:491–500; Vorhees CV, Schaefer TL, Williams MT (2007) Synapse 61:488–499]. Lack of differences may be the result of compensatory mechanisms in reaction to a constitutive knock out of Pet-1 or 5-HT may not be as important in learning and memory as previously suspected.

Elucidation of the ELK1 target gene network reveals a role in the coordinate regulation of core components of the gene regulation machinery

Transcription factors play an important role in orchestrating the activation of specific networks of genes through targeting their proximal promoter and distal enhancer regions. However, it is unclear how the specificity of downstream responses is maintained by individual members of transcription-factor families and, in most cases, what their target repertoire is. We have used ChIP-chip analysis to identify the target genes of the ETS-domain transcription factor ELK1. Two distinct modes of ELK1 target gene selection are identified; the first involves redundant promoter binding with other ETS-domain family members; the second occurs through combinatorial binding with a second transcription factor SRF, which specifies a unique group of target genes. One of the most prominent groups of genes forming the ELK1 target network includes classes involved in core gene expression control, namely, components of the basal transcriptional machinery, the spliceosome and the ribosome. Amongst the set of genes encoding the basal transcription machinery components, are a functionally linked subset of GTFs and TAFs. Our study, therefore, reveals an unsuspected level of coordinate regulation of components of the core gene expression control machinery and also identifies two different modes of promoter targeting through binding with a second transcription factor or redundant binding with other ETS-domain family members.

The Ets-Domain Transcription Factor Spdef Promotes Maturation of Goblet and Paneth Cells in the Intestinal Epithelium

Stem cells within the intestinal epithelium generate daughter cells that undergo lineage commitment and maturation through the combined action of the Wnt and Notch signaling cascades. Both pathways, in turn, regulate transcription factor networks that further define differentiation toward either enterocytes or 1 of 3 secretory cell lineages (Paneth, goblet, or enteroendocrine cells). In this study, we investigated the role of the Wnt-responsive, Ets-domain transcription factor Spdef in the differentiation of goblet and Paneth cells. The in vivo function of Spdef was examined by disrupting the Spdef gene in mice (Spdef(-/-) mice) and analyzing the intestinal phenotype using a range of histologic techniques and DNA microarray profiling. In accordance with expression data, we found that loss of Spdef severely impaired the maturation of goblet and Paneth cells and, conversely, led to an accumulation of immature secretory progenitors. Spdef appears to positively and negatively regulate a specific subset of goblet and Paneth cell genes, including Cryptdins, Mmp7, Ang4, Kallikreins, and Muc2. Spdef acts downstream of Math1 to promote terminal differentiation of a secretory progenitor pool into Paneth and goblet cells.

Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Signaling Initiates a Dynamic Interplay between Sumoylation and Ubiquitination To Regulate the Activity of the Transcriptional Activator PEA3

Many transcription factors are controlled through SUMO modification, and in the majority of cases this modification results in enhancements in their repressive properties. In some instances, SUMO modification and its associated repressive activities can be reversed by the action of intracellular signaling pathways, leading to enhanced transcriptional capacities of transcription factors. Here we have investigated sumoylation of the ETS domain transcription factor PEA3 and its interplay with the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. PEA3 is modified by SUMO in vitro and in vivo on multiple sites in its N-terminal region. Activation of the ERK MAP kinase pathway promotes sumoylation of PEA3. Importantly, sumoylation of PEA3 is required for maximal activation of target gene promoters, including MMP-1 and COX-2. Molecularly, sumoylation is selectively required for synergistic activation of target gene expression with the coactivator CBP. Moreover, sumoylation of PEA3 is required for ubiquitination of PEA3 and promotes its degradation, suggesting that SUMO-mediated recycling of PEA3 plays a role in PEA3-mediated promoter activation. Thus, in contrast to the majority of other transcription factors studied, sumoylation of PEA3 plays a positive role in PEA3-mediated transcriptional activation and the ERK MAP kinase pathway cooperates with rather than antagonizes this process.

Fgf-Dependent Etv4/5 Activity Is Required for Posterior Restriction of Sonic hedgehog and Promoting Outgrowth of the Vertebrate Limb

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. Here, we show that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development.

Suppression Subtractive Hybridization Analysis of Gene Expression during Late Kidney Development Identifies the Developmentally Regulated Gene rPEA3

Background: While early kidney development has been studied exhaustively, the later stages of nephrogenesis that occur after birth in the rodent are relatively poorly understood. To gain insight into this process, we detected the alterations in gene expression in rat kidney at two postnatal stages, P0 (0 day after birth), the time at which nephrogensis is still active, and P21 (21 days after birth), when nephrogenesis is complete. Methods: Sprague-Dawley rats were mated, and appearance of a vaginal plug was designated as E0. Kidneys were dissected from embryos at E13, E15, E17 and E19, and from postnatal days P0, P7, P14, P21 and adult rats. Suppression subtractive hybridization (SSH) analysis was performed and highly expressed genes were evaluated as molecular markers by real-time reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunofluorescence, and Western blot. Results: Several differentially expressed genes were identified, including rPEA3, a member of the PEA3 subfamily of Ets domain transcription factors. Real time RT-PCR analysis revealed that rPEA3 exhibited dynamic developmental regulation, with high levels of expression from embryonic day E15 until birth, and declining levels thereafter. By in situ hybridization, rPEA3 mRNA was detected in the ureteric bud (UB) and surrounding metanephric mesenchyme of the kidneys from E15 until birth, but was undetectable in mature kidneys. Double-immunofluorescence staining showed that both rPEA3 and WT1 expressed in the condensed mesenchymal cells at E15 and E17; and later from E19 to P7, both expressed in the epithelial cells of ureteric bud and their branches. Conclusions: These studies provide compelling evidence that SSH is an effective method for identifying genes that are regulated during renal development, and suggest that the newly identified gene rPEA3 may play an important role in kidney development and differentiation.

Opposite Effects of Interleukin-1α and Transforming Growth Factor-β2 Induce Stage-Specific Regulation of Junctional Adhesion Molecule-B Gene in Sertoli Cells

In the mammalian testis, junctional adhesion molecule-B (JAM-B) is found at the blood-testis barrier between Sertoli cells and the apical ectoplasmic specializations between Sertoli and germ cells. The expression of JAM-B is tightly regulated to allow the transit of developing germ cells across the blood-testis barrier and the timely release of mature spermatids at stage VIII. In this study, the basal transcription of JAM-B in the mouse Sertoli cell line, MSC-1 cells, was examined. We found that the constitutive expression of JAM-B is carried out by the binding of specificity proteins (Sps), ETS domain transcription factor Elk-1 (Elk1), neuron-restrictive silencer factor (NRSF), and E2F transcription factor 3 (E2F3) to various cis-acting elements including TG interacting factor (TGIF), Elk-1, NRSF, and proximal Sp1 (pSp1) + E2F binding motifs. We also investigated the effects of two cytokines IL-1alpha and TGF-beta2 on JAM-B expression. IL-1alpha promotes JAM-B expression by facilitating the binding of Elk-1 to TGIF and pSp1 + E2F motifs in a p38-dependent manner, which leads to an additive effect on Sp1- and NRSF-mediated JAM-B transactivation. TGF-beta2 inhibits JAM-B transcription via the activation of mothers against decapentaplegic (Smad) proteins and activated Smads compete with specificity proteins (Sp1 and Sp3) for the TGIF motif, resulting in JAM-B repression. IL-1alpha and Smad3 expression have been reported to be stage specific. IL-1alpha is absent in the seminferous epithelium at stages VII-VIII, whereas a high level of nuclear Smad3 level is found at the same stages. This study shows for the first time that IL-1alpha and TGF-beta2 regulate JAM-B expression in an opposite manner, and in vitro data obtained herein provide some clues on how junctions are regulated in the testis.

Elk-1 interacts with neuronal microtubules and relocalizes to the nucleus upon phosphorylation

ETS domain transcription factor Elk-1 has been primarily studied in the regulation of genes in response to mitogenic stimuli, however the presence of Elk-1 in axonal projections of largely post-mitotic adult hippocampal sections has been reported. This finding has initially led us to a basic question: how is Elk-1 anchored to neuronal projections? To that end, we have investigated the intracellular localization of Elk-1 and its biochemical interactions with neuronal microtubules in model systems. Our results showed co-localization of Elk-1 with microtubules in hippocampal cultures and SH-SY5Y neuroblastoma cell lines, and have further demonstrated that Elk-1 protein can biochemically interact with microtubules in vitro. Analysis of the protein sequence has indicated many putative microtubule binding domains, with the strongest binding prediction in amino acids 314–325, and our results show that Elk-1 can bind to microtubules through most of these regions, but no interaction was observed through the DNA binding domain, where no putative binding motifs were predicted. We further show that upon serum induction, most of the phospho-Elk-1 translocates to the nucleus, which is independent of translation. We propose that Elk-1 is anchored to neuronal microtubules in resting or unstimulated cells, and upon stimulation is phosphorylated, which relocalizes phospho-Elk-1 to the nucleus in neurons.

Genome-Wide Analysis of the Zebrafish ETS Family Identifies Three Genes Required for Hemangioblast Differentiation or Angiogenesis

ETS domain transcription factors have been linked to hematopoiesis, vasculogenesis, and angiogenesis. However, their biological functions and the mechanisms of action, remain incompletely understood. Here, we have performed a systematic analysis of zebrafish ETS domain genes and identified 31 in the genome. Detailed gene expression profiling revealed that 12 of them are expressed in blood and endothelial precursors during embryonic development. Combined with a phylogenetic tree assay, this suggests that some of the coexpressed genes may have redundant or additive functions in these cells. Loss-of-function analysis of 3 of them, erg, fli1, and etsrp, demonstrated that erg and fli1 act cooperatively and are required for angiogenesis possibly via direct regulation of an endothelial cell junction molecule, VE-cadherin, whereas etsrp is essential for primitive myeloid/endothelial progenitors (hemangioblasts) in zebrafish. Taken together, these results provide a global view of the ETS genes in the zebrafish genome during embryogenesis and provide new insights on the functions and biology of erg, fli1, and etsrp, which could be applicable to higher vertebrates, including mice and humans.

Wildtype Elk-1, but not a SUMOylation mutant, represses egr-1 expression in SH-SY5Y neuroblastomas

Elk-1, an ETS domain transcription factor of the TCF (ternary complex factor) subfamily, is known to be involved in the regulation of immediate-early genes such as c-fos upon mitogen activation, and thus commonly implied in cell proliferation. Early growth response-1, Egr-1, which was known to be an immediate-early gene, has recently been shown to be pro-apoptotic for SH-SY5Y neuroblastoma cells. In that respect, it was not clear whether Elk-1 would activate or repress from this promoter, since Elk-1 is mostly associated with proliferation and not apoptosis. In this study, we wanted to address whether Elk-1 activates or represses egr-1 promoter in neuroblastoma cells, and using a combination of RNA interference and reporter analyses, we present evidence that egr-1 gene is repressed by Elk-1 in normally cycling SH-SY5Y neuroblastoma cell line in a SUMO (small ubiquitin-related modifier)-dependent manner, a potential mechanism used by these cells to bypass apoptosis.

Raf signaling but not the ERK effector SAP-1 is required for regulatory T cell development.

Regulatory T cells (T(reg)) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and T(reg) development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, T(reg) development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4(+) cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3(+) and Foxp3(-) CD4(+) populations. In SAP-1-deficient CD4(+)CD25(+) T(reg) cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4(+)CD25(+) T(reg) cells to suppress CD4(+)CD25(-) cell proliferation in vitro. Finally the suppressive activity of CD4(+)CD25(+) T(reg) cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of T(reg) cells in the thymus are thus distinct from those required for "conventional" T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of T(reg) cells.

GA-Binding Protein Is Dispensable for Neuromuscular Synapse Formation and Synapse-Specific Gene Expression

The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and other "synaptic" genes is required for synapse-specific transcription. The Ets domain transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression. Inactivation of GABPalpha, the DNA-binding subunit of GABP, leads to early embryonic lethality, preventing analysis of synapse formation in gabpalpha mutant mice. To study the role of GABP at neuromuscular synapses, we conditionally inactivated gabpalpha in skeletal muscle and studied synaptic differentiation and muscle gene expression. Although expression of rb, a target of GABP, is elevated in muscle tissue deficient in GABPalpha, clustering of synaptic AChRs at synapses and synapse-specific gene expression are normal in these mice. These data indicate that GABP is dispensable for synapse-specific transcription and maintenance of normal AChR expression at synapses.

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions.

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

Tamoxifen-induced activation of p21Waf1/Cip1 gene transcription is mediated by Early Growth Response-1 protein through the JNK and p38 MAP kinase/Elk-1 cascades in MDA-MB-361 breast carcinoma cells

Tamoxifen (TAM) is a synthetic non-steroidal anti-estrogen compound that is widely used as an effective chemotherapeutic agent for treatment and prevention of breast cancer. Unfortunately, prolonged treatment with TAM causes TAM-responsive tumors to become TAM resistant through an as-yet-unknown mechanism. To develop novel anti-breast cancer agents that are therapeutically superior to TAM, we must first fully understand the biological effects of TAM. In this study, we found that TAM treatment of MDA-MB-361 breast cancer cells activated p21Waf1/Cip1 gene transcription independently of p53. Furthermore, TAM-induced p21Waf1/Cip1 promoter activity was enhanced by transient expression of the gene encoding Early Growth Response-1 (Egr-1) protein, a transcription factor that plays an important role in cell growth and differentiation. The TAM-induced p21Waf1/Cip1 promoter activity was blocked by the expression of small interfering RNA (siRNA) targeted to Egr-1 mRNA. In addition, induction of Egr-1 expression by TAM occurred at the transcriptional level via Ets-domain transcription factor Elk-1 through the JNK and p38 mitogen-activated protein (MAP) kinase pathways. Inhibition of the JNK and p38 MAP kinase signals inhibited Egr-1-mediated p21Waf1/Cip1 promoter activity. We conclude that TAM stimulation of p21Waf1/Cip1 gene transcription in MDA-MB-361 cells depends largely on Elk-1-mediated Egr-1 expression induced by activation of the JNK and p38 MAP kinase pathways.