The Role of Eph Receptors and Ephrins in Corneal Physiology and Diseases.

The cornea, while appearing to be simple tissue, is actually an extremely complex structure. In order for it to retain its biomechanical and optical properties, perfect organization of its cells is essential. Proper regeneration is especially important after injuries and in the course of various diseases. Eph receptors and ephrin are mainly responsible for the proper organization of tissues as well as cell migration and communication. In this review, we present the current state of knowledge on the role of Eph and ephrins in corneal physiology and diseases, in particular, we focused on the functions of the epithelium and endothelium. Since the role of Eph and ephrins in the angiogenesis process has been well established, we also analyzed their influence on conditions with corneal neovascularization.

Role of forward and reverse signaling in Eph receptor and ephrin mediated cell segregation.

Eph receptor and ephrin signaling has a major role in segregating distinct cell populations to form sharp borders. Expression of interacting Ephs and ephrins typically occurs in complementary regions, such that polarised activation of both components occurs at the interface. Forward signaling through Eph receptors can drive cell segregation, but it is unclear whether reverse signaling through ephrins can also contribute. We have tested the role of reverse signaling, and of polarised versus non-polarised activation, in assays in which contact repulsion drives cell segregation and border sharpening. We find that polarised forward signaling drives stronger segregation than polarised reverse signaling. Nevertheless, reverse signaling contributes since bidirectional Eph and ephrin activation drives stronger segregation than unidirectional forward signaling alone. In contrast, non-polarised Eph activation drives little segregation. We propose that although polarised forward signaling is the principal driver of segregation, reverse signaling enables bidirectional repulsion which prevents mingling of each population into the other.

Dying to communicate: apoptotic functions of Eph/Ephrin proteins.

The Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors constitute the largest family of receptor tyrosine kinases and interact with a group of ligands called Ephrins. An essential feature of the Eph receptors and Ephrin ligands is that both are membrane-bound and, upon cell-cell interaction, initiate a bidirectional signaling involving both the receptor (forward signaling) and the ligand (reverse signaling). They regulate a large set of pleiotropic functions in virtually every tissue and physiological system. In vitro as well as in vivo data support a role for Eph and Ephrin molecules in cellular processes such as proliferation, cell-cell attraction and repulsion, motility and sorting. An increasing amount of evidence supports a role for these molecules in apoptosis and, although this function in cell death has been barely examined, the available information warrants a global consideration, to identify unmet needs and potential research avenues. Here we propose a comprehensive analysis of the data available regarding the importance of Ephs and Ephrins in cell death mechanisms throughout a large array of physiological systems.

[Regulatory Effects of Erythropoietin-producing Hepatocyte Kinase Receptor and Ephrin Ligand on Bone Remodeling].

During the process of bone remodeling,the bone homeostasis is tightly controlled by the coupling of bone resorption and bone formation,depending upon cellular communication between osteoclasts and osteoblasts. Many studies have identified that the bi-directional transduction of erythropoietin producing hepatocyte kinase receptor and ephrin ligand (Eph/ephrin) is one of signal transduction pathways in bone remodeling. This review focus on the potential role of Eph/ephrin in bone remodeling,especially in alveolar remodeling.

α2-chimaerin is required for Eph receptor-class-specific spinal motor axon guidance and coordinate activation of antagonistic muscles.

Axonal guidance involves extrinsic molecular cues that bind growth cone receptors and signal to the cytoskeleton through divergent pathways. Some signaling intermediates are deployed downstream of molecularly distinct axon guidance receptor families, but the scope of this overlap is unclear, as is the impact of embryonic axon guidance fidelity on adult nervous system function. Here, we demonstrate that the Rho-GTPase-activating protein α2-chimaerin is specifically required for EphA and not EphB receptor signaling in mouse and chick spinal motor axons. Reflecting this specificity, the loss of α2-chimaerin function disrupts the limb trajectory of extensor-muscle-innervating motor axons the guidance of which depends on EphA signaling. These embryonic defects affect coordinated contraction of antagonistic flexor-extensor muscles in the adult, indicating that accurate embryonic motor axon guidance is critical for optimal neuromuscular function. Together, our observations provide the first functional evidence of an Eph receptor-class-specific intracellular signaling protein that is required for appropriate neuromuscular connectivity.

Eph and ephrin signaling: lessons learned from spinal motor neurons.

In nervous system assembly, Eph/ephrin signaling mediates many axon guidance events that shape the formation of precise neuronal connections. However, due to the complexity of interactions between Ephs and ephrins, the molecular logic of their action is still being unraveled. Considerable advances have been made by studying the innervation of the limb by spinal motor neurons, a series of events governed by Eph/ephrin signaling. Here, we discuss the contributions of different Eph/ephrin modes of interaction, downstream signaling and electrical activity, and how these systems may interact both with each other and with other guidance molecules in limb muscle innervation. This simple model system has emerged as a very powerful tool to study this set of molecules, and will continue to be so by virtue of its simplicity, accessibility and the wealth of pioneering cellular studies.

Eph/ephrin signaling in epidermal differentiation and disease.

Eph receptor tyrosine kinases mediate cell-cell communication by interacting with ephrin ligands residing on adjacent cell surfaces. In doing so, these juxtamembrane signaling complexes provide important contextual information about the cellular microenvironment that helps orchestrate tissue morphogenesis and maintain homeostasis. Eph/ephrin signaling has been implicated in various aspects of mammalian skin physiology, with several members of this large family of receptor tyrosine kinases and their ligands present in the epidermis, hair follicles, sebaceous glands, and underlying dermis. This review focuses on the emerging role of Eph receptors and ephrins in epidermal keratinocytes where they can modulate proliferation, migration, differentiation, and death. The activation of Eph receptors by ephrins at sites of cell-cell contact also appears to play a key role in the maturation of intercellular junctional complexes as keratinocytes move out of the basal layer and differentiate in the suprabasal layers of this stratified, squamous epithelium. Furthermore, alterations in the epidermal Eph/ephrin axis have been associated with cutaneous malignancy, wound healing defects and inflammatory skin conditions. These collective observations suggest that the Eph/ephrin cell-cell communication pathway may be amenable to therapeutic intervention for the purpose of restoring epidermal tissue homeostasis and integrity in dermatological disorders.

Eph and ephrin signaling in the formation of topographic maps.

The axonal connections between the retina and its midbrain target, the superior colliculus (SC), is mapped topographically, such that the spatial relationships of cell bodies in the retina are maintained when terminating in the SC. Topographic map development uses a Cartesian mapping system such that each axis of the retina is mapped independently. Along the nasal-temporal mapping axis, EphAs and ephrin-As, are graded molecular cues required for topographic mapping while the dorsal-ventral axis is mapped in part via EphB and ephrin-Bs. Because both Ephs and ephrins are cell surface molecules they can signal in the forward and reverse directions. Eph/ephrin signaling leads to changes in cytoskeletal dynamics that lead to actin depolymerization and endocytosis guiding axons via attraction and repulsion.

Eph/ephrin interactions modulate muscle satellite cell motility and patterning.

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. We have recently shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. We show here that, in vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, we propose that it might also play a role in satellite cell-mediated muscle repair. Therefore, we investigated whether Eph-ephrin signaling would produce changes in satellite cell directional motility. Using a classical ephrin 'stripe' assay, we found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. We hypothesize that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle.

The role of EPH receptors in cancer-related epithelial-mesenchymal transition.

Erythropoietin-producing hepatoma (EPH) receptors are considered the largest family of receptor tyrosine kinases and play key roles in physiological and pathologic processes in development and disease. EPH receptors are often overexpressed in human malignancies and are associated with poor prognosis. However, the functions of EPH receptors in epithelial-mesenchymal transition (EMT) remain largely unknown. This review depicts the relationship between EPH receptors and the EMT marker E-cadherin as well as the crosstalk between EPH receptors and the signaling pathways involved EMT. Further discussion is focused on the clinical significance of EPH receptors as candidates for targeting in cancer therapeutics. Finally, we summarize how targeted inhibition of both EPH receptors and EMT-related signaling pathways represents a novel strategy for cancer treatment.

Ankyrin repeat-rich membrane spanning protein (kidins220) is required for neurotrophin and ephrin receptor-dependent dendrite development.

Dendrites are the primary sites on neurons for receiving and integrating inputs from their presynaptic partners. Defects in dendrite development perturb the formation of neural circuitry and impair information processing in the brain. Extracellular cues are important for shaping the dendritic morphogenesis, but the underlying molecular mechanisms are not well understood. In this study, we examined the role of ARMS (ankyrin repeat-rich membrane spanning protein), also known as Kidins220 (kinase D-interacting substrate of 220 kDa), previously identified as a downstream target of neurotrophin and ephrin receptors, in dendrite development. We report here that knockdown of ARMS/Kidins220 by in utero electroporation impairs dendritic branching in mouse cerebral cortex, and silencing of ARMS/Kidins220 in primary rat hippocampal neurons results in a significant decrease in the length, number, and complexity of the dendritic arbors. Overexpression of cell surface receptor tyrosine kinases, including TrkB and EphB2, in ARMS/Kidins220-deficient neurons can partially rescue the defective dendritic phenotype. More importantly, we show that PI3K (phosphoinositide-3-kinase)- and Akt-mediated signaling pathway is crucial for ARMS/Kidins220-dependent dendrite development. Furthermore, loss of ARMS/Kidins220 significantly reduced the clustering of EphB2 receptor signaling complex in neurons. Our results collectively suggest that ARMS/Kidins220 is a key player in organizing the signaling complex to transduce the extracellular stimuli to cellular responses during dendrite development.

The ephrin signaling pathway regulates morphology and adhesion of mouse granulosa cells in vitro.

Follicle-stimulating hormone (FSH)-mediated changes in granulosa cell adhesion and morphology are essential for preovulatory follicle development, given the dramatic changes in follicle size and granulosa cell number that occur during this transition. Members of the Eph-ephrin family of cell-positioning and adhesion molecules, a family that consists of ephrin ligands and their Ephrin (Eph) receptors, regulate cell location, adhesion, and migration during embryonic development and tumor growth. However, very little is known about ephrin signaling during folliculogenesis. We have found that FSH increases the expression of several members of the Eph-ephrin family and that this signaling regulates granulosa cell morphology and adhesion. FSH induced increased mRNA levels of the ephrin ligand, ephrin-A5 (Efna5), and its receptors, Eph receptors A3, A5, and A8 (Epha3, Epha5, and Epha8, respectively), in granulosa cells. Immunofluorescence studies indicated that EFNA5 and EPHA5 are located in the membrane of granulosa cells of developing mouse follicles. Eph-ephrin signaling directly affected granulosa cell morphology and adhesion. Recombinant EFNA5 reduced cell spreading and increased cell rounding in mouse primary granulosa cells and in a rat granulosa cell line, whereas EPHA5 reduced granulosa cell adhesion in both model systems. Both FSH and forskolin also increased Efna5 and Epha5 mRNA levels in rat and human granulosa cell lines, indicating that FSH regulates these genes via the cAMP-dependent protein kinase A pathway and that this regulation is conserved across different species. The present study identifies Eph-ephrin signaling as a novel FSH-mediated pathway regulating granulosa cell morphology and adhesion.

Ephs and ephrins resurface in inflammation, immunity, and atherosclerosis.

Despite significant advancements in treatment regimens, cardiovascular disease remains a worldwide leader of morbidity, mortality, and healthcare cost. A large percentage of cardiovascular disease is directly attributable to the process of atherosclerosis, a chronic inflammatory disease of the vessel wall. In the hunt for novel therapeutic targets in cardiovascular disease, neuronal guidance molecules are emerging as significant regulators of cardiovascular remodeling and inflammation. The Eph family of neuronal guidance molecules comprises the largest family of receptor tyrosine kinases in the mammalian genome. While best characterized in embryogenesis and carcinogenesis, Eph receptors and their ephrin ligands are becoming increasingly recognized as important players in chronic inflammatory diseases and immune function. Herein we discuss the current evidence for how Eph/ephrin interactions, particularly EphA2/ephrinA1 and EphB/ephrinB2, affect inflammation and cardiovascular disease.

The role of endocytosis in activating and regulating signal transduction.

Endocytosis is increasingly understood to play crucial roles in most signaling pathways, from determining which signaling components are activated, to how the signal is subsequently transduced and/or terminated. Whether a receptor-ligand complex is internalized via a clathrin-dependent or clathrin-independent endocytic route, and the complexes' subsequent trafficking through specific endocytic compartments, to then be recycled or degraded, has profound effects on signaling output. This review discusses the roles of endocytosis in three markedly different signaling pathways: the Wnt, Notch, and Eph/Ephrin pathways. These offer fundamentally different signaling systems: (1) diffusible ligands inducing signaling in one cell, (2) membrane-tethered ligands inducing signaling in a contacting receptor cell, and (3) bi-directional receptor-ligand signaling in two contacting cells. In each of these systems, endocytosis controls signaling in fascinating ways, and comparison of their similarities and dissimilarities will help to expand our understanding of endocytic control of signal transduction across multiple signaling pathways.

Eph-dependent cell-cell adhesion and segregation in development and cancer.

Numerous studies attest to essential roles for Eph receptors and their ephrin ligands in controlling cell positioning and tissue patterning during normal and oncogenic development. These studies suggest multiple, sometimes contradictory, functions of Eph-ephrin signalling, which under different conditions can promote either spreading and cell-cell adhesion or cytoskeletal collapse, cell rounding, de-adhesion and cell-cell segregation. A principle determinant of the balance between these two opposing responses is the degree of receptor/ligand clustering and activation. This equilibrium is likely altered in cancers and modulated by somatic mutations of key Eph family members that have emerged as candidate cancer markers in recent profiling studies. In addition, cross-talk amongst Ephs and with other signalling pathways significantly modulates cell-cell adhesion, both between and within Eph- and ephrin-expressing cell populations. This review summarises our current understanding of how Eph receptors control cell adhesion and morphology, and presents examples demonstrating the importance of these events in normal development and cancer.

Critical role of the axonal guidance cue EphrinB2 in lung growth, angiogenesis, and repair.

RATIONALE: Lung diseases characterized by alveolar damage currently lack efficient treatments. The mechanisms contributing to normal and impaired alveolar growth and repair are incompletely understood. Axonal guidance cues (AGC) are molecules that guide the outgrowth of axons to their targets. Among these AGCs, members of the Ephrin family also promote angiogenesis, cell migration, and organogenesis outside the nervous system. The role of Ephrins during alveolar growth and repair is unknown. OBJECTIVES: We hypothesized that EphrinB2 promotes alveolar development and repair. METHODS: We used in vitro and in vivo manipulation of EphrinB2 signaling to assess the role of this AGC during normal and impaired lung development. MEASUREMENTS AND MAIN RESULTS: In vivo EphrinB2 knockdown using intranasal siRNA during the postnatal stage of alveolar development in rats arrested alveolar and vascular growth. In a model of O(2)-induced arrested alveolar growth in newborn rats, air space enlargement, loss of lung capillaries, and pulmonary hypertension were associated with decreased lung EphrinB2 and receptor EphB4 expression. In vitro, EphrinB2 preserved alveolar epithelial cell viability in O(2), decreased O(2)-induced alveolar epithelial cell apoptosis, and accelerated alveolar epithelial cell wound healing, maintained lung microvascular endothelial cell viability, and proliferation and vascular network formation. In vivo, treatment with intranasal EphrinB2 decreased alveolar epithelial and endothelial cell apoptosis, preserved alveolar and vascular growth in hyperoxic rats, and attenuated pulmonary hypertension. CONCLUSION: The AGC EphrinB2 may be a new therapeutic target for lung repair and pulmonary hypertension.

Ephrin-B reverse signaling controls septation events at the embryonic midline through separate tyrosine phosphorylation-independent signaling avenues.

We report that the disruption of bidirectional signaling between ephrin-B2 and EphB receptors impairs morphogenetic cell-cell septation and closure events during development of the embryonic midline. A novel role for reverse signaling is identified in tracheoesophageal foregut septation, as animals lacking the cytoplasmic domain of ephrin-B2 present with laryngotracheoesophageal cleft (LTEC), while both EphB2/EphB3 forward signaling and ephrin-B2 reverse signaling are shown to be required for midline fusion of the palate. In a third midline event, EphB2/EphB3 are shown to mediate ventral abdominal wall closure by acting principally as ligands to stimulate ephrin-B reverse signaling. Analysis of new ephrin-B2(6YFΔV) and ephrin-B2(ΔV) mutants that specifically ablate ephrin-B2 tyrosine phosphorylation- and/or PDZ domain-mediated signaling indicates there are at least two distinct phosphorylation-independent components of reverse signaling. These involve both PDZ domain interactions and a non-canonical SH2/PDZ-independent form of reverse signaling that may utilize associations with claudin family tetraspan molecules, as EphB2 and activated ephrin-B2 molecules are specifically co-localized with claudins in epithelia at the point of septation. Finally, the developmental phenotypes described here mirror common human midline birth defects found with the VACTERL association, suggesting a molecular link to bidirectional signaling through B-subclass Ephs and ephrins.

Control of cell adhesion and compartmentalization in the intestinal epithelium.

Continuous cell renewal in the intestinal mucosa occurs without disrupting the integrity of the epithelial layer. Despite the restrictions imposed by strong cell-to-cell adhesions, epithelial intestinal cells migrate constantly between tissue compartments. Alterations in cell adhesion and compartmentalization play key roles in diseases of the intestine. In particular, decreased E-cadherin-mediated adhesion during inflammatory bowel disease and loss of EphB/ephrin-B-mediated compartmentalization in colorectal cancer have recently emerged as key players of these prevalent pathologies. Here we will review our current knowledge on how cell-to-cell adhesion, migration and cell positioning are coordinated in the intestinal epithelium. We will highlight what the in vivo genetic analysis of intestinal epithelium has taught us about the complex regulation of cell adhesion and migration in homeostasis and disease.

Signal transduction pathways participating in homeostasis and malignant transformation of the intestinal tissue.

Intestinal homeostasis is a complex and tightly regulated process governed by a variety of signalling pathways that balance cell proliferation and differentiation. As revealed by extensive use of defined mouse models, perturbations within the signalling circuitry trigger initial expansion of premalignant cells. In this review, we attempt to summarise recent advances in the knowledge of the cellular signalling mechanisms that drive tumorigenesis in the human and mouse intestine.

Eph receptors and ephrins in neuron-astrocyte communication at synapses.

Neuron-glia communication is essential for regulating the properties of synaptic connections in the brain. Astrocytes, in particular, play a critical and complex role in synapse development, maintenance, and plasticity. Likewise, neurons reciprocally influence astrocyte physiology. However, the molecular signaling events that enable astrocytes and neurons to effectively communicate with each other are only partially defined. Recent findings have revealed that Eph receptor tyrosine kinases and ephrins play an important role in contact-dependent neuron-glia communication at synapses. Upon binding, these two families of cell surface-associated proteins trigger bidirectional signaling events that regulate the structural and physiological properties of both neurons and astrocytes. This review will focus on the emerging role of Eph receptors and ephrins in neuron-astrocyte interaction at synapses and discuss implications for synaptic plasticity, behavior, and disease.