A potential role for Eph receptor signalling during migration of corneal endothelial cells.

The corneal endothelium is a monolayer of epithelial cells that lines the posterior surface of the cornea and is essential for maintenance of corneal transparency. Wound healing within the corneal endothelium typically occurs through cell spreading and migration rather than through proliferation. The mechanisms that control corneal endothelial cell migration are unclear. In this study we demonstrate that cultures of corneal endothelial cells display reduced migration in scratch wound assays, and reduced levels of E-cadherin mRNA, following suppression of ligand-activated Eph receptor signalling by treatment with lithocholic acid. Two Eph receptors, EphA1 and EphA2, were subsequently detected in corneal endothelial cells, and their potential involvement during migration was explored through gene silencing using siRNAs. EphA2 siRNA reduced levels of mRNA for both EphA2 and N-cadherin, but increased levels of mRNA for both EphA1 and E-cadherin. No effect, however, was observed for EphA2 siRNA on migration. Our results indicate a potential role for Eph receptor signalling during corneal endothelial cell migration via changes in cadherin expression. Nevertheless, defining a precise role for select Eph receptors is likely to be complicated by crosstalk between Eph-mediated signalling pathways.

The role of Eph/ephrin molecules in stromal­hematopoietic interactions.

Bone marrow mesenchymal stromal/stem cells(BMSC) are fundamental regulatory elements of the hematopoietic stem cell niche; however, the molecular signals that mediate BMSC support of hematopoiesis are poorly understood. Recent studies indicate that BMSC and hematopoietic stem/progenitors cells differentially express the Eph cell surface tyrosine kinase receptors, and their ephrinligands. Eph/ephrin interactions are thought to mediate cross-talk between BMSC and different hematopoietic cell populations to influence cell development, migration and function. This review summarizes Eph/ephrin interactions in the regulation of BMSC communication with hematopoietic stem/progenitor cells and discusses Eph/ephrintargeted therapeutic strategies that are currently being pursued or various hematotological malignancies.

Eph receptors and their ligands: promising molecular biomarkers and therapeutic targets in prostate cancer.

Although at present, there is a high incidence of prostate cancer, particularly in the Western world, mortality from this disease is declining and occurs primarily only from clinically significant late stage tumors with a poor prognosis. A major current focus of this field is the identification of new biomarkers which can detect earlier, and more effectively, clinically significant tumors from those deemed "low risk", as well as predict the prognostic course of a particular cancer. This strategy can in turn offer novel avenues for targeted therapies. The large family of Receptor Tyrosine Kinases, the Ephs, and their binding partners, the ephrins, has been implicated in many cancers of epithelial origin through stimulation of oncogenic transformation, tumor angiogenesis, and promotion of increased cell survival, invasion and migration. They also show promise as both biomarkers of diagnostic and prognostic value and as targeted therapies in cancer. This review will briefly discuss the complex roles and biological mechanisms of action of these receptors and ligands and, with regard to prostate cancer, highlight their potential as biomarkers for both diagnosis and prognosis, their application as imaging agents, and current approaches to assessing them as therapeutic targets. This review demonstrates the need for future studies into those particular family members that will prove helpful in understanding the biology and potential as targets for treatment of prostate cancer.

The role of Eph receptors in lens function and disease.

Cataract is the single largest contributor to blindness in the world, with the disease having a strong genetic component. In recent years the Eph family of receptor tyrosine kinases has been identified as a key regulator in lens clarity. In this review we discuss the roles of the Eph receptors in lens biology and cataract development.

EphrinA1 stimulates cell attachment and inhibits cell aggregation through the EphA receptor pathway in human endometrial carcinoma-derived Ishikawa cells.

BACKGROUND: Recently, the Eph-ephrinA system was proposed to contribute to the initial interaction between the maternal endometrial epithelium and embryonic trophectoderm. Since the Eph-ephrin interaction can induce adhesive and/or repulsive forces into the cells, we examined the possible role of this system in functional changes in endometrial epithelial cells using endometrial carcinoma-derived Ishikawa cells. METHODS: The expressions of EphA1, A2 and A4 on Ishikawa cells were examined by RT-PCR and western blotting analyses. The effects of recombinant ephrinA1 on Ishikawa cells were also examined by western blot analysis and cell attachment and aggregation assays. RESULTS: EphA1, A2 and A4 were expressed on Ishikawa cells. Recombinant ephrinA1 bound to the surfaces of Ishikawa cells and induced phosphorylation of EphA2 and A4. In bovine serum albumin-blocked nitrocellulose-coated dishes, Ishikawa cells remained floating and aggregated with each other. Under these conditions, immobilized ephrinA1 promoted Ishikawa cell attachment with increased tyrosine phosphorylation in focal adhesion kinase. In addition, immobilized ephrinA1 reversibly inhibited Ishikawa cell aggregation. Gene-reduction of EphA1, A2 and A4 by siRNAs attenuated the inhibitory effects of ephrinA1 on cell aggregation, confirming that ephrinA1 affects Ishikawa cell functions through Eph-ephrinA interaction. CONCLUSIONS: This study demonstrated that the Eph-ephrinA system can promote cell attachment along with intercellular dissociation in Ishikawa cells. These findings suggest that this system can induce functional changes in endometrial epithelial cells.

The role of Eph receptors and ephrin ligands in colorectal cancer.

Eph receptors and their ephrin ligands constitute the largest subfamily of receptor tyrosine kinases and are components of the cell signaling pathways involved during development. Eph and ephrin overexpression have been documented in a variety of human cancers including gastrointestinal malignancies and in particular colorectal malignancies. EphB and ephrin B proteins have been implicated in the homeostasis of the gastrointestinal tract where EphB2- and EphB3-ephrin B signaling regulates cell sorting in the mature epithelium. These proteins are also reported to be upregulated in colon carcinomas. The EphA/ephrin A system has also been implicated in epithelial tissue structure and function. More recently, EphA receptors and their corresponding ligands have been implicated in numerous malignancies. Of these, EphA2 in particular has been intensively investigated and has been proposed as a therapeutic target. An interesting observation emerging from these studies is the role for Ephs and ephrins in critical aspects of cell adhesion, migration and positioning, and a crucial role in tumor progression and metastasis. However, the underlying role of Ephs and ephrins in these processes has generally been studied on individual Eph or ephrin genes. Given the multiplicity of Eph expression on gut epithelial cells, a more global approach is needed to define the precise role of Eph-ephrin interaction in malignant transformation. Here, we will review the recent advances on the role of Eph-ephrin signaling in colorectal malignancies.

A presynaptic homeostatic signaling system composed of the Eph receptor, ephexin, Cdc42, and CaV2.1 calcium channels.

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release remain largely unknown. In a screen, we isolated mutations in Drosophila ephexin (Rho-type guanine nucleotide exchange factor) that disrupt the homeostatic enhancement of presynaptic release following impairment of postsynaptic glutamate receptor function at the Drosophila neuromuscular junction. We show that Ephexin is sufficient presynaptically for synaptic homeostasis and localizes in puncta throughout the nerve terminal. However, ephexin mutations do not alter other aspects of neuromuscular development, including morphology or active zone number. We then show that, during synaptic homeostasis, Ephexin functions primarily with Cdc42 in a signaling system that converges upon the presynaptic CaV2.1 calcium channel. Finally, we show that Ephexin binds the Drosophila Eph receptor (Eph) and Eph mutants disrupt synaptic homeostasis. Based on these data, we propose that Ephexin/Cdc42 couples synaptic Eph signaling to the modulation of presynaptic CaV2.1 channels during the homeostatic enhancement of presynaptic release.

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice.

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.

Eph, a protein family coming of age: more confusion, insight, or complexity?

Since the mid-1980s, Eph receptors have evolved from being regarded as orphan receptors with unknown functions and ligands to becoming one of the most complex "global positioning systems" that regulates cell traffic in multicellular organisms. During this time, there has been an exponentially growing interest in Ephs and ephrin ligands, coinciding with important advances in the way biological function is interrogated through mapping of genomes and manipulation of genes. As a result, many of the original concepts that used to define Eph signaling and function went overboard. Clearly, the need for progress in understanding Eph-ephrin biology and the underlying molecular principles involved has been compelling. Many cell-positioning programs during normal and oncogenic development-in particular, the patterning of skeletal, vascular, and nervous systems-are modulated in some way by Eph-ephrin function. Undeniably, the complexity of the underlying signaling networks is considerable, and it seems probable that systems biology approaches are required to further improve our understanding of Eph function.

Bidirectional signaling of ErbB and Eph receptors at synapses.

Synapse development and remodeling are regulated by a plethora of molecules such as receptor tyrosine kinases (RTKs), a family of cell surface receptors that play critical roles in neural development. Two families of RTKs implicated in synaptic functions, ErbBs and Ephs, share similar characteristics in terms of exhibiting forward and reverse signaling. In this review, we will discuss the latest advances in the functions of ErbBs and Ephs at the synapse, including dendritic spine morphogenesis, synapse formation and maturation, and synaptic transmission and plasticity. In addition to signaling at interneuronal synapses, communication between neuron and glia is increasingly implicated in the control of synaptic functions. Studies on RTKs and their cognate ligands in glial cells enhance our understanding on the nature of 'tripartite synapse'. Implications of these signaling events in human diseases will be discussed.

EphA and ephrin-A proteins regulate integrin-mediated T lymphocyte interactions.

Progressive interaction with other cells is critical to all aspects of T-cell biology - from migration through tissues, to recognition of antigen-presenting cells. We demonstrate a novel mechanism for regulating T lymphocyte adhesion and thus cell-cell interactions, showing that T-cell-expressed EphA and ephrin-A proteins not only regulate adhesive properties, but do so in a diametrically opposing fashion. Integrin-mediated adhesion is stimulated by ephrin-A activation, while EphA signalling is inhibitory. Increasing ephrin-A expression enhances T-cell interactions not only with purified integrin ligands but also endothelial cells, while EphA activation down-regulates these interactions. In accordance with an in vivo role for these proteins in regulating cell-cell interactions, activation of ephrin-A signalling was found to alter the trafficking of injected T lymphocytes to peripheral lymph nodes in vivo. Given the ubiquitous nature of EphA/ephrin-A expression in tissues, these proteins likely play a significant role in regulating T-cell interactions.

Regulation of angiogenesis by Eph-ephrin interactions.

The large families of Eph receptor tyrosine kinases and their ephrin ligands transduce signals in a cell-cell contact-dependent fashion and thereby coordinate the growth, differentiation, and patterning of almost every organ and tissue. Eph-ephrin interactions can trigger a wide array of cellular responses, including cell adhesion, boundary formation, and repulsion. The exact mechanisms leading to this diversity of responses are unclear but appear to involve differential signaling, proteolytic cleavage of ephrins, and endocytosis of the ligand-receptor complex. In the developing cardiovascular system, Eph and ephrin molecules control the angiogenic remodeling of blood vessels and lymphatic vessels and play essential roles in endothelial cells as well as in supporting pericytes and vascular smooth muscle cells. Recent evidence suggests that Ephs and ephrins may also be involved in pathological angiogenesis, in particular, the neovascularization of tumors. Consequently, the expression, interactions, or signaling of Eph-ephrin molecules might be targets for future therapeutic approaches.

Eph receptor and ephrin signaling in developing and adult brain of the honeybee (Apis mellifera).

Roles for Eph receptor tyrosine kinase and ephrin signaling in vertebrate brain development are well established. Their involvement in the modulation of mammalian synaptic structure and physiology is also emerging. However, less is known of their effects on brain development and their function in adult invertebrate nervous systems. Here, we report on the characterization of Eph receptor and ephrin orthologs in the honeybee, Apis mellifera (Am), and their role in learning and memory. In situ hybridization for mRNA expression showed a uniform distribution of expression of both genes across the developing pupal and adult brain. However, in situ labeling with Fc fusion proteins indicated that the AmEphR and Amephrin proteins were differentially localized to cell body regions in the mushroom bodies and the developing neuropiles of the antennal and optic lobes. In adults, AmEphR protein was localized to regions of synaptic contacts in optic lobes, in the glomeruli of antennal lobes, and in the medial lobe of the mushroom body. The latter two regions are involved in olfactory learning and memory in the honeybee. Injections of EphR-Fc and ephrin-Fc proteins into the brains of adult bees, 1 h before olfactory conditioning of the proboscis extension reflex, significantly reduced memory 24 h later. Experimental amnesia in the group injected with ephrin-Fc was apparent 1 h post-training. Experimental amnesia was also induced by post-training injections with ephrin-Fc suggesting a role in recall. This is the first demonstration that Eph molecules function to regulate the formation of memory in insects.

Drosophila Eph receptor guides specific axon branches of mushroom body neurons.

The conserved Eph receptors and their Ephrin ligands regulate a number of developmental processes, including axon guidance. In contrast to the large vertebrate Eph/Ephrin family, Drosophila has a single Eph receptor and a single Ephrin ligand, both of which are expressed within the developing nervous system. Here, we show that Eph and Ephrin can act as a functional receptor-ligand pair in vivo. Surprisingly, and in contrast to previous results using RNA-interference techniques, embryos completely lacking Eph function show no obvious axon guidance defects. However, Eph/Ephrin signaling is required for proper development of the mushroom body. In wild type, mushroom body neurons bifurcate and extend distinct branches to different target areas. In Eph mutants, these neurons bifurcate normally, but in many cases the dorsal branch fails to project to its appropriate target area. Thus, Eph/Ephrin signaling acts to guide a subset of mushroom body branches to their correct synaptic targets.

Eph receptor tyrosine kinases in angiogenesis: from development to disease.

Angiogenesis, the process by which new blood vessels sprout and branch from existing vasculature, is crucial for vascular remodeling during embryogenesis and in normal tissue homeostasis, such as in the female reproductive tract. Angiogenesis can also contribute to the pathogenesis of diseases such as cancer and retinopathy. The Eph family of receptor tyrosine kinases and their ligands, called ephrins, has emerged as critical regulators of vascular remodeling in the embryo. More recently, these molecules have been associated with post-natal angiogenic remodeling and tumor neovascularization. This review provides an overview of recent advances in our understanding of Eph/ephrins in angiogenesis, with an emphasis on development and disease, and the potential for targeting these molecules in anti-angiogenic therapy.

Multiple roles of ephrins during the formation of thalamocortical projections: maps and more.

The functional architecture of the cerebral cortex is based on intrinsic connections that precisely link neurons from distinct cortical laminae as well as layer-specific afferent and efferent projections. Experimental strategies using in vitro assays originally developed by Friedrich Bonhoeffer have suggested that positional cues confined to individual layers regulate the assembly of local cortical circuits and the formation of thalamocortical projections. One of these wiring molecules is ephrinA5, a ligand for Eph receptor tyrosine kinases. EphrinA5 and Eph receptors exhibit highly dynamic expression patterns in distinct regions of the cortex and thalamus during early and late stages of thalamocortical and cortical circuit formation. In vitro assays suggest that ephrinA5 is a multifunctional wiring molecule for different populations of cortical and thalamic axons. Additionally, the expression patterns of ephrinA5 during cortical development are consistent with this molecule regulating, in alternative ways, specific components of thalamic and cortical connectivity. To test this directly, the organization of thalamocortical projections was examined in mice lacking ephrinA5 gene expression. The anatomical studies in ephrinA5 knockout animals revealed a miswiring of limbic thalamic projections and changes in neocortical circuits that were predicted from the expression pattern and the in vitro analysis of ephrinA5 function.

Eph receptor deficiencies lead to altered cochlear function.

Ephrins and Eph receptors are a family of molecules that have been implicated in many developmental processes including neuronal network formation, guidance of cell migration, and axonal pathfinding. These molecules exhibit the ability to send bidirectional signals following ligand-receptor interactions resulting from cell-cell contacts. Gene-targeted knockout mice of B-class ephrins and Eph receptors have been shown to display phenotypic responses that correlate with anatomical defects. For example, disruption of the EphB2 receptor leads to defects of the vestibular system, including pathfinding abnormalities in efferent axons and reduced endolymph production. Such developmental distortions lead to deficiencies in ionic homeostasis and repetitive circling behaviors. The present study demonstrates that B-class ephrins and Eph receptors are expressed in cochlear tissues, suggesting that they may play some role in auditory function. To determine whether ephrins and Eph receptors have a functional role in the peripheral auditory system, distortion-product otoacoustic emission (DPOAE) levels, collected across a broad frequency range, were compared between groups of mice expressing different Eph receptor genotypes. In particular, EphB1 and EphB3 receptor knockout mice exhibited significantly diminished DPOAE levels as compared to wild-type littermates, indicating that these specific Eph receptors are necessary for normal cochlear function.

An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans.

During sexual reproduction in most animals, oocytes arrest in meiotic prophase and resume meiosis (meiotic maturation) in response to sperm or somatic cell signals. Despite progress in delineating mitogen-activated protein kinase (MAPK) and CDK/cyclin activation pathways involved in meiotic maturation, it is less clear how these pathways are regulated at the cell surface. The Caenorhabditis elegans major sperm protein (MSP) signals oocytes, which are arrested in meiotic prophase, to resume meiosis and ovulate. We used DNA microarray data and an in situ binding assay to identify the VAB-1 Eph receptor protein-tyrosine kinase as an MSP receptor. We show that VAB-1 and a somatic gonadal sheath cell-dependent pathway, defined by the CEH-18 POU-class homeoprotein, negatively regulate meiotic maturation and MAPK activation. MSP antagonizes these inhibitory signaling circuits, in part by binding VAB-1 on oocytes and sheath cells. Our results define a sperm-sensing control mechanism that inhibits oocyte maturation, MAPK activation, and ovulation when sperm are unavailable for fertilization. MSP-domain proteins are found in diverse animal taxa, where they may regulate contact-dependent Eph receptor signaling pathways.