| Abstract |
ERF (Ets-2 Repressor Factor) is a transcriptional repressor that belongs to ETS family and is regulated by RTK/RAS/ERK signalling pathway. ETS family members control important biological processes, including cell proliferation, differentiation, apoptosis, hematopoiesis, angiogenesis and oncogenesis. Respectively, ERF could have such a variety of actions, thinking that Erf gene is ubiquitously expressed in the developing mouse embryo and adult tissues as well as in all cell lines that have been examined. ERF’s ERK-mediated phosphorylation determines its subcellular localization. After mitogenic stimulation ERF is phosphorylated by ERK and exported from the nucleus to the cytoplasm, while in the absence of mitogenic stimulation, ERF is accumulating in the nucleus in a non-phosphorylated state and thus can act as a transcriptional repressor, regulating the expression of a big diversity of genes. Although much is known about Erf regulation and some of its basic functions, such as cell cycle arrest by direct suppression of c-Myc with a Rb-dependent manner, as well as inhibition of ets- and ras- induced transformation and Ewing’s sarcoma in cellular systems, with this work we advanced the ERF study a step further. Specifically, here we examined the ERF function to epithelial-mesenchymal transition (EMT) and mammary oncogenesis, as well as the differentiation of trophoblast stem cells (TSCs) that is a critical process in the formation of placenta.
Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-β (TGF-β) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF) for its
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ability to interfere with TGF-β–induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-β–induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-β signaling through Smads was mostly unaffected, and ERF suppressed the TGF-β–induced EMT via Semaphorin-7a repression. Inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-β–induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-β–signaling pathways. The cause of these findings, it was to start the study of Erf deletion from the mouse mammary glands or the mouse primary mammary epithelial cells (PMECs), thus associating ERF with mammary oncogenesis. However, this study is in progress, so there are still no any safe conclusions.
Furthermore, we examined the ERF role in the differentiation of trophoblast stem cells (TSCs) that contribute to the placenta formation, a process in which EMT procedure that we studied initially is very important. Homozygous deletion of the Erf in mice has been shown to block chorionic trophoblast differentiation leading to the failure of chorioallantoic fusion and embryo death. Fibroblast growth factor (FGF) signaling is important for the proper trophoblast stem cell (TSC) differentiation and development of the hemochorial placenta. Lack of Fgf2 promotes TSC differentiation while FGF4 or FGF2 is required for murine TSC maintenance. Employing molecular and cellular approaches we show here that low levels of Fgf2 mRNA can be detected ex vivo in TSC. This expression is repressed via direct interaction of ERF with the Fgf2 transcription unit, is increased in the absence of ERF and is decreased in the presence of an ERF mutation resistant to ERK phosphorylation. Fgf2 inhibition by ERF appears to be necessary for the proper differentiation of the chorionic trophoblast stem cells and may account for the Erf knock out phenotype. Finally, differentiation of ERF overexpressing TSC lines suggests that in addition to Fgf2 mediated effects Erf may have an additional role in the commitment of chorionic trophoblasts towards syncytiotrophoblast.
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Finally, it was shown that reduced dosage of ERF causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. So within this wider work, here using chromatin immunoprecipitation in mouse embryonic fibroblasts (MEFs) and high-throughput sequencing (ChIP-seq), we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.
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