The role of glial and neuronal Eph/ephrin signaling in Drosophila mushroom body development and sleep and circadian behavior.

The Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species.

The first identification of complete Eph-ephrin signalling in ctenophores and sponges reveals a role for neofunctionalization in the emergence of signalling domains.

BACKGROUND: Animals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores - non-bilaterian basal animals - typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected. RESULTS: Here, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported. CONCLUSIONS: We propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.

Rescue and characterization of recombinant cedar virus, a non-pathogenic Henipavirus species.

BACKGROUND: Hendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses. METHODS: We constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay. RESULTS: Successful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3. CONCLUSIONS: The recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.

Overexpression of Receptor Tyrosine Kinase EphB4 Triggers Tumor Growth and Hypoxia in A375 Melanoma Xenografts: Insights from Multitracer Small Animal Imaging Experiments.

Experimental evidence has associated receptor tyrosine kinase EphB4 with tumor angiogenesis also in malignant melanoma. Considering the limited in vivo data available, we have conducted a systematic multitracer and multimodal imaging investigation in EphB4-overexpressing and mock-transfected A375 melanoma xenografts. Tumor growth, perfusion, and hypoxia were investigated by positron emission tomography. Vascularization was investigated by fluorescence imaging in vivo and ex vivo. The approach was completed by magnetic resonance imaging, radioluminography ex vivo, and immunohistochemical staining for blood and lymph vessel markers. Results revealed EphB4 to be a positive regulator of A375 melanoma growth, but a negative regulator of tumor vascularization. Resulting in increased hypoxia, this physiological characteristic is considered as highly unfavorable for melanoma prognosis and therapy outcome. Lymphangiogenesis, by contrast, was not influenced by EphB4 overexpression. In order to distinguish between EphB4 forward and EphrinB2, the natural EphB4 ligand, reverse signaling a specific EphB4 kinase inhibitor was applied. Blocking experiments show EphrinB2 reverse signaling rather than EphB4 forward signaling to be responsible for the observed effects. In conclusion, functional expression of EphB4 is considered a promising differentiating characteristic, preferentially determined by non-invasive in vivo imaging, which may improve personalized theranostics of malignant melanoma.

Investigation of the interactions between the EphB2 receptor and SNEW peptide variants.

EphB2 interacts with cell surface-bound ephrin ligands to relay bidirectional signals. Overexpression of the EphB2 receptor protein has been linked to different types of cancer. The SNEW (SNEWIQPRLPQH) peptide binds with high selectivity and moderate affinity to EphB2, inhibiting Eph-ephrin interactions by competing with ephrin ligands for the EphB2 high-affinity pocket. We used rigorous free energy perturbation (FEP) calculations to re-evaluate the binding interactions of SNEW peptide with the EphB2 receptor, followed by experimental testing of the computational results. Our results provide insight into dynamic interactions of EphB2 with SNEW peptide. While the first four residues of the SNEW peptide are already highly optimized, change of the C-terminal end of the peptide has the potential to improve SNEW-binding affinity. We identified a PXSPY motif that can be similarly aligned with several other EphB2-binding peptides.

A Pharmacological Investigation of Eph-Ephrin Antagonism in Prostate Cancer: UniPR1331 Efficacy Evidence.

The Eph kinases are the largest receptor tyrosine kinases (RTKs) family in humans. PC3 human prostate adenocarcinoma cells are a well-established model for studying Eph-ephrin pharmacology as they naturally express a high level of EphA2, a promising target for new cancer therapies. A pharmacological approach with agonists did not show significant efficacy on tumor growth in prostate orthotopic murine models, but reduced distal metastasis formation. In order to improve the comprehension of the pharmacological targeting of Eph receptors in prostate cancer, in the present work, we investigated the efficacy of Eph antagonism both in vitro and in vivo, using UniPR1331, a small orally bioavailable Eph-ephrin interaction inhibitor. UniPR1331 was able to inhibit PC3 cells' growth in vitro in a dose-dependent manner, affecting the cell cycle and inducing apoptosis. Moreover, UniPR1331 promoted the PC3 epithelial phenotype, downregulating epithelial mesenchymal transition (EMT) markers. As a consequence, UniPR1331 reduced in vitro PC3 migration, invasion, and vasculomimicry capabilities. The antitumor activity of UniPR1331 was confirmed in vivo when administered alone or in combination with cytotoxic drugs in PC3-xenograft mice. Our results demonstrated that Eph antagonism is a promising strategy for inhibiting prostate cancer growth, especially in combination with cytotoxic drugs.

Signals transduced by Eph receptors and ephrin ligands converge on MAP kinase and AKT pathways in human cancers.

Eph receptors, the largest known family of receptor tyrosine kinases, and ephrin ligands have been implicated in a variety of human cancers. The novel bidirectional signaling events initiated by binding of Eph receptors to their cognate ephrin ligands modulate many cellular processes such as proliferation, metastasis, angiogenesis, invasion, and apoptosis. The relationships between the abundance of a unique subset of Eph receptors and ephrin ligands with associated cellular processes indicate a key role of these molecules in tumorigenesis. The combinatorial expression of these molecules converges on MAP kinase and/or AKT/mTOR signaling pathways. The intracellular target proteins of the initial signal may, however, vary in some cancers. Furthermore, we have also described the commonality of up- and down-regulation of individual receptors and ligands in various cancers. The current state of research in Eph receptors illustrates MAP kinase and mTOR pathways as plausible targets for therapeutic interventions in various cancers.

Artesunate-induced Cellular Effects Are Mediated by Specific EPH Receptors and Ephrin Ligands in Breast Carcinoma Cells.

BACKGROUND/AIM: The aberrant regulation of erythropoietin-producing hepatocellular carcinoma (EPH) receptors and ephrin ligands has been implicated in breast carcinoma, and artesunate has been shown to have anticancer effects. The aim of this study was to characterize the involvement of EPH receptors and ephrin ligands in mediating artesunate (ART)-induced growth suppression of normal breast cells and breast carcinoma cell lines. MATERIALS AND METHODS: The normal breast epithelial cells (MCF10A), non-invasive ductal breast carcinoma cells (MCF7), and invasive triple-negative breast carcinoma cells (MDA-MB-231) were grown in the absence or the presence of different concentrations of artesunate. The cells were counted, and total RNA was isolated. The abundance of transcripts corresponding to EPH receptors and ephrin ligands was determined by quantitative polymerase chain reaction. RESULTS: Cell viability was significantly reduced when cells were treated with artesunate, with MDA-MB-231 cells having the highest sensitivity. Artesunate had no significant effect on transcription of EPH/ephrins in MCF10A cells, but markedly increased EPHA8, EPHA10, EPHB6 and ephrin-A2 expression in MCF7 cells, and significantly increased EPHA3 and EPHA10 expression while reducing that of EPHA7 and ephrin-A3 in MDA-MB-231 cells. CONCLUSION: The relative changes in artesunate-treated MCF7 and MDA-MB-231 cells as compared to similarly treated MCF10A cells allow us to implicate combinatorial expression and receptor interactions for EPH receptor-mediated signal transduction that converges into pathways responsible for cell growth, proliferation, and apoptosis. Specifically, the alterations in EPHA7, EPHA8, EPHA10 and EPHB6 transcripts appear to be important participants in artesunate-mediated cellular effects.

Stem-like Cells from Invasive Breast Carcinoma Cell Line MDA-MB-231 Express a Distinct Set of Eph Receptors and Ephrin Ligands.

BACKGROUND/AIM: Breast cancer cell lines consist of bulk tumor cells and a small proportion of stem-like cells. While the bulk cells are known to express a distinct combination of Eph receptors and ephrin ligands, the transcript profiles of stem-like cells in these cell lines have not been adequately characterized. The aim of this study was to determine Eph receptor/ephrin ligand profiles of cancer stem cells specific to a triple negative breast carcinoma cell line. MATERIALS AND METHODS: The normal breast cell line MCF10A and the invasive breast carcinoma cell line MDA-MB-231 were used to isolate CD24+/CD24- cell populations. The profiles of Eph receptors and ephrin ligands were determined by real-time PCR and the relative abundance in bulk and stem cells were compared. RESULTS: Based on the mean ΔCT values, the descending order of abundance was as follows. Ephrin-A5 > EPHA2 > (EPHA8, EPHB2) > ephrin-B2 > (EPHA7, EPHB4, ephrin-A4) > ephrin-A3 > ephrin-A1 > (EPHB3, ephrin-B1) > EPHA4 > EPHA1 > EPHA10. EPHA6 and ephrin-A2 transcripts were not detectable in stem cells from either cell line. The expression of EPHA4, EPHA7, EPHA8, and ephrin-A5 in MDA-MB-231 stem cells was up-regulated by 12, 20, ~500, and 6.5-fold respectively. CONCLUSION: The up-regulation of transcripts for EPHA8 and its cognate ligand, ephrin-A5, in the stem cells isolated from MDA-MB-231, suggest their involvement in the invasiveness of this cell line. Based on literature reports, we propose the role of EPHA8 and ephrin-A5 in MDA-MB-231 stem cells via the PI3K-AKT-mTOR pathway.

Progress in Researches of Eph/ephrin Signaling in Coupling of Bone Resorption and Bone Formation / 生物化学与生物物理进展

Osteoclastic bone resorption and osteoblastic bone formation are coordinated as a coupled mechanism to effect the development of bone and to maintain bone homeostasis. Recently reported Eph/ephrin bidirectional signaling between osteoclasts and osteoblasts plays a pivol role in bone homeostasis and casts new light on coupling of bone resorption and bone formation, which is gaining more and more attention in researches of bone biology and bone diseases. The present article aims to address the researches on the Eph/ephrin bidirectional signaling between osteoblasts and osteoclasts with molecular constitution, mechanism of the signal transduction, biological significance and so on.

Advancement in studies on Eph family proteins / 中国药理学通报

The Eph protein family,consisting of Eph receptors and their corresponding membrane-anchored protein ligands-Ephrin,is no doubt the largest receptor protein tyrosine kinases family till now.Interactions between Eph receptors and Ephrin ligands based on their special structure might make the Eph protein family one of new targets of disease′s treatment and consequently studies related to Eph proteins and their receptors have draw more and more attention.This overview will focus on recent progresses in the classification,gene expression,protein structure,function and protein-receptor interactions of the Eph protein family and their receptors.Much of the focus of this overview is on their physiological and pathophysiological role in nervous system and their therapeutic perspective from several aspects.