The Eph receptor/ephrin system: an emerging player in the invasion game

Eph receptor tyrosine kinases (Ephs) and their membrane-anchored ligands (ephrins) form a vital cell communication system capable of bi-directional signaling. This Eph receptor/ephrin system has classically been demonstrated to play a role in development. However, emerging evidence has revealed differential expression of Ephs and ephrins in numerous cancers. Recent studies suggest that this system influences invasive behaviour, promoting a more aggressive and metastatic phenotype. Hence, this minireview summarizes the current understanding of the contribution of both Eph receptors and their ephrin ligands to invasiveness in cancer, as well as their use as potential therapeutic targets.     

Eph receptors and their ephrin ligands are expressed in developing mouse pancreas

Pancreas development involves branching morphogenesis concomitantly to differentiation of endocrine, exocrine and ductal cell types from a single population of pancreatic precursors. These processes depend on many signals and factors that also control development of the central nervous system. In the latter, Eph receptors and their class-A (GPI-anchored) and class-B (transmembrane) ephrin ligands control cell migration and axon-pathfinding, help establish regional patterns and act as labels for cell positioning. This raised the question as to whether and where Ephs and ephrins are expressed during pancreas development. Here we have identified the Eph and ephrin genes that are expressed in mouse embryonic pancreas, as detected by RT-PCR analysis. In situ hybridization experiments showed that Ephs and ephrins are mainly expressed in the burgeoning structures of the epithelium which differentiate into exocrine acini. Binding experiments on whole pancreas demonstrated the presence of functional Eph receptors. They showed that EphBs are expressed by the pancreatic epithelium at embryonic day (e) 12.5 and that, from e14.5 on, Ephs of both classes are expressed by the pancreatic epithelium and then become restricted to developing acini. We conclude that specific members of the Eph/ephrin family are expressed in embryonic pancreas according to a dynamic temporal and regional pattern.     

Eph-modulated cell morphology, adhesion and motility in carcinogenesis

Eph receptor tyrosine kinases (Ephs) and their membrane anchored ephrin ligands (ephrins) form an essential cell-cell communication system that directs the positioning, adhesion and migration of cells and cell layers during development. While less prominent in normal adult tissues, there is evidence that up-regulated expression and de-regulated function of Ephs and ephrins in a large variety of human cancers may promote a more aggressive and metastatic tumour phenotype. However, in contrast to other RTKs, Ephs do not act as classical proto-oncogenes and do not effect cell proliferation or differentiation. Mounting evidence suggests that Eph receptors, through de-regulated re-emergence of their mode of action in the embryo may direct cell movements and positioning during metastasis, invasion and tumour angiogenesis. This review discusses these and other emerging roles of Eph receptors during oncogenesis.     

Membrane‐mediated regulation of vascular identity

Vascular diseases span diverse pathology, but frequently arise from aberrant signaling attributed to specific membrane‐associated molecules, particularly the Eph‐ephrin family. Originally recognized as markers of embryonic vessel identity, Eph receptors and their membrane‐associated ligands, ephrins, are now known to have a range of vital functions in vascular physiology. Interactions of Ephs with ephrins at cell‐to‐cell interfaces promote a variety of cellular responses such as repulsion, adhesion, attraction, and migration, and frequently occur during organ development, including vessel formation. Elaborate coordination of Eph‐ and ephrin‐related signaling among different cell populations is required for proper formation of the embryonic vessel network. There is growing evidence supporting the idea that Eph and ephrin proteins also have postnatal interactions with a number of other membrane‐associated signal transduction pathways, coordinating translation of environmental signals into cells. This article provides an overview of membrane‐bound signaling mechanisms that define vascular identity in both the embryo and the adult, focusing on Eph‐ and ephrin‐related signaling. We also discuss the role and clinical significance of this signaling system in normal organ development, neoplasms, and vascular pathologies. Birth Defects Research (Part C) 108:65–84, 2016. © 2016 Wiley Periodicals, Inc.     

Expression and function of the Eph A receptors and their ligands ephrins A in the rat thymus

Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.     

Eph receptors at synapses: implications in neurodegenerative diseases

Precise regulation of synapse formation, maintenance and plasticity is crucial for normal cognitive function, and synaptic failure has been suggested as one of the hallmarks of neurodegenerative diseases. In this review, we describe the recent progress in our understanding of how the receptor tyrosine kinase Ephs and their ligands ephrins regulate dendritic spine morphogenesis, synapse formation and maturation, as well as synaptic plasticity. In particular, we discuss the emerging evidence implicating that deregulation of Eph/ephrin signaling contributes to the aberrant synaptic functions associated with cognitive impairment in Alzheimer's disease. Understanding how Eph/ephrin regulates synaptic function may therefore provide new insights into the development of therapeutic agents against neurodegenerative diseases.     

Ectodomain structures of Eph receptors

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication that regulate axon guidance, long-term potentiation, and stem cell development, among others. By now, many Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Since both the receptor and the ligand are membrane-bound, their interaction leads to the multimerization of both molecules to distinct clusters within their respective plasma membranes, resulting in the formation of discrete signaling centers. In addition, and unique to Eph receptors and ephrins, their interaction initiates bi-directional signaling cascades where information is transduced in the direction of both the receptor- and the ligand-bearing cells. The Ephs and the ephrins are divided into two subclasses, A and B, based on their affinities for each other and on sequence conservation. Crystal structures and other biophysical studies have indicated that isolated extracellular Eph and ephrin domains initially form high-affinity heterodimers around a hydrophobic loop of the ligand that is buried in a hydrophobic pocket on the surface of the receptor. The dimers can then further arrange by weaker interactions into higher-order Eph/ephrin clusters observed in vivo at the sites of cell-cell contact. Although the hetero-dimerization is a universal way to initiate signaling, other extracellular domains of Ephs are involved in the formation of higher-order clusters. The structures also show important differences defining the unique partner preferences of the two ligand and receptor subclasses, namely, how subclass specificity is determined both by individual interacting residues and by the precise architectural arrangement of ligands and receptors within the complexes.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation.     

Homotypic receptor-receptor interactions regulating Eph signaling

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, and mediate many other cell-cell communication events. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. Recent structural, biochemical and cell-biological studies revealed that these assemblies are generated not only via Eph-ephrin interactions, but also via homotypic interactions between neighboring receptor molecules. In addition, Eph-Eph interactions mediate receptor pre-clustering, which ensures fast and efficient activation once ligands come into contact range. Here we summarize the current knowledge about the homotypic Eph-Eph interactions and discuss how they could modulate the initiation of Eph/ephrin signaling.     

Homotypic receptor-receptor interactions regulating Eph signaling

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, and mediate many other cell-cell communication events. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. Recent structural, biochemical and cell-biological studies revealed that these assemblies are generated not only via Eph-ephrin interactions, but also via homotypic interactions between neighboring receptor molecules. In addition, Eph-Eph interactions mediate receptor pre-clustering, which ensures fast and efficient activation once ligands come into contact range. Here we summarize the current knowledge about the homotypic Eph-Eph interactions and discuss how they could modulate the initiation of Eph/ephrin signaling.     

Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph-Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Profiling Eph receptor expression in cells and tissues: A targeted mass spectrometry approach

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.     

Looking forward to EphB signaling in synapses

Eph receptors and their ligands ephrins comprise a complex signaling system with diverse functions that span a wide range of tissues and developmental stages. The variety of Eph receptor functions stems from their ability to mediate bidirectional signaling through trans-cellular Eph/ephrin interactions. Initially thought to act by directing repulsion between cells, Ephs have also been demonstrated to induce and maintain cell adhesive responses at excitatory synapses in the central nervous system. EphB receptors are essential to the development and maintenance of dendritic spines, which accommodate the postsynaptic sites of most glutamatergic excitatory synapses in the brain. Functions of EphB receptors are not limited to control of the actin cytoskeleton in dendritic spines, as EphB receptors are also involved in the formation of functional synaptic specializations through the regulation of glutamate receptor trafficking and functions. In addition, EphB receptors have recently been linked to the pathophysiology of Alzheimer's disease and neuropathic pain, thus becoming promising targets for therapeutic interventions. In this review, we discuss recent findings on EphB receptor functions in synapses, as well as the mechanisms of bidirectional trans-synaptic ephrin-B/EphB receptor signaling that shape dendritic spines and influence post-synaptic differentiation.