Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

In silico prediction of interaction between Nipah virus attachment glycoprotein and host cell receptors Ephrin-B2 and Ephrin-B3 in domestic and peridomestic mammals.

Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between -8.0 to -19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.

Activation of spinal ephrin-B3/EphBs signaling induces hyperalgesia through a PLP-mediated mechanism.

Ephrin B/EphB signaling pathway is involved in the regulation of pain caused by spinal cord injury. However, the role of ephrin-B3/EphBs signaling in regulation of nociceptive information is poorly understood. In the present study, formalin-induced inflammatory pain, mechanical allodynia and thermal hyperalgesia, was measured using Efnb3 mutant mice (Efnb3-/- ) and wild-type (Efnb3+/+ ) mice. The spinal cord (L4-6) was selected for molecular and cellular identification by western blotting and immunofluorescence. Efnb3 mutant mice showed a significant increased the thermal and mechanical threshold, followed by aberrant thin myelin sheath. Furthermore, expression of proteolipid protein (PLP) was significantly lower in L4-6 spinal cord of Efnb3-/- mice. These morphological and behavioral abnormalities in mutant mice were rescued by conditional knock-in of wild-type ephrin-B3. Intrathecal administration of specific PLP siRNA significantly increased the thermal and mechanical threshold hyperalgesia in wild-type mice. However, overexpressing PLP protein by AAV9-PLP could decrease the sensitivity of mice to thermal and mechanical stimuli in Efnb3-/- mice, compared with scrabble Efnb3-/- mice. Further, Efnb3lacz mice, which have activities to initiate forward signaling, but transduce reverse signals by ephrin-B3, shows normal acute pain behavior, compared with wild type mice. These findings indicate that a key molecule Efnb3 act as a prominent contributor to hyperalgesia and essential roles of ephrin-B3/EphBs in nociception through a myelin-mediated mechanism.

Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein.

Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiVB) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.

A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation.

The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.

Structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by Cedar virus.

Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.

Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling.

Schwann cells (SC) enter the central nervous system (CNS) in pathophysiological conditions. However, how SC invade the CNS to remyelinate central axons remains undetermined. We studied SC migratory behavior ex vivo and in vivo after exogenous transplantation in the demyelinated spinal cord. The data highlight for the first time that SC migrate preferentially along blood vessels in perivascular extracellular matrix (ECM), avoiding CNS myelin. We demonstrate in vitro and in vivo that this migration route occurs by virtue of a dual mode of action of Eph/ephrin signaling. Indeed, EphrinB3, enriched in myelin, interacts with SC Eph receptors, to drive SC away from CNS myelin, and triggers their preferential adhesion to ECM components, such as fibronectin via integrinß1 interactions. This complex interplay enhances SC migration along the blood vessel network and together with lesion-induced vascular remodeling facilitates their timely invasion of the lesion site. These novel findings elucidate the mechanism by which SC invade and contribute to spinal cord repair.

Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs.

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.

Eph/ephrin signalling serves a bidirectional role in lipopolysaccharide­induced intestinal injury.

A growing body of evidence has demonstrated that Eph/ephrin signalling may serve a central role in intestinal diseases. However, whether erythropoietin­producing hepatocellular (Eph)/ephrin signalling is associated with the development of post­infectious irritable bowel syndrome (PI­IBS) is still unknown. In the present study, the role of Eph/Ephrin signalling in lipopolysaccharide (LPS)­induced intestinal injury was evaluated in vivo and in vitro. LPS treatment significantly increased the levels of proinflammatory mediators [monocyte chemoattractant protein­1, tumour necrosis factor α, interleukin (IL)­1ß, IL­6, intercellular adhesion molecule 1 and vascular cell adhesion molecule­1], activated the EphA2­Ephrin A1, protein kinase B (Akt)­nuclear factor (NF)­κB, Src­NF­κB and Wnt/ß­catenin signalling pathways, and inhibited EphB1­Ephrin B3 signalling in colon tissues, and primary cultured enteric neuronal and glial cells. Notably, EphA2 monoclonal antibody (mAb) treatment or Ephrin B3 overexpression could partially alleviate the LPS­induced upregulation of proinflammatory mediators, and Akt­NF­κB, Src­NF­κB and Wnt/ß­catenin signalling pathways. In addition, EphA2 mAb treatment could partially inhibit LPS­induced inactivation of EphB­Ephrin B3 signalling, while Ephrin B3 overexpression could abrogate LPS­induced activation of EphA2­Ephrin A1 signalling. EphB1/Ephrin B3 signalling may antagonise the EphA2/Ephrin A1­dependent pathway following LPS treatment. The results associated with the EphA2 signaling pathway, indicated that Eph/ephrin signalling may serve a bidirectional role in LPS­induced intestinal injury. Eph/ephrin signalling may be a novel therapeutic target for LPS­induced intestinal injury and potentially PI­IBS.

Ephrin-B3 binds both cell-associated and secreted proteoglycans.

The ephrin family of membrane proteins binds Eph tyrosine kinase receptors. We have previously shown that ephrin-B3 also binds to heparan sulfate proteoglycans (HSPGs). We now show that ephrin-B3 can bind both secretory and cell associated PGs, such as agrin, collagen XVIII, Perlecan, and CD44, and indicate that such interaction with cell associated PGs involves a complex including 20 and 45 kDa proteins. Ephrin-B3 binding to HEK-293T cells is blocked by a secretory variant of CD44 (v3-v10), while over-expression of membrane associated CD44 increased ephrin-B3 binding. In addition, ephrin-B3 precipitated CD44 expressed by the oral squamous carcinoma cell line H376. Moreover, ephrin-B3 binding affinities to heparin and CD44 in solution was strong. In conclusion, we have identified secretory and cell associated PGs with high ability to bind ephrin-B3 and suggest that ephrin-B3 can bind to a protein complex organized by a membrane associated PG.

Ephrin­b3 modulates hippocampal neurogenesis and the reelin signaling pathway in a pilocarpine­induced model of epilepsy.

Ephrin­B3 is important in the regulation of cell proliferation, differentiation and migration via cell­cell contact, and can activate the reelin pathway during brain development. However, the effect of ephrin­B3 on hippocampal neurogenesis and the reelin pathway in epilepsy remains to be fully elucidated. In the present study, the expression of ephrin­B3 in pilocarpine­induced status epilepticus (SE) rats was investigated. SYBR Green­based reverse transcription­quantitative polymerase chain reaction analysis, immunohistochemical labeling and western blot analysis were used to detect the gene and protein expression levels of ephrin­B3 and reelin pathway proteins. Immunofluorescence staining of doublecortin (DCX) was utilized to analyze hippocampal neurogenesis. The data revealed that the mRNA and protein expression levels of ephrin­B3 in the hippocampus decreased during the spontaneous seizure period. Of note, the expression of reelin and its downstream phosphorylation disabled 1 (p­Dab1) were also notably decreased during the spontaneous seizure period, which showed similar dynamic changes as in the expression of ephrin­B3. In addition, it was found that the number of DCX­labeled neuronal progenitor cells was increased in the hippocampus following pilocarpine­induced SE. To further clarify the role of ephrin­B3 in neurogenesis and the reelin pathway in epilepsy, an exogenous ephrin­B3 clustering stimulator, EphB3­Fc, was infused into the bilateral hippocampus of the rats post­SE. Following EphB3­Fc injection, it was found that the expression levels of reelin and p­Dab1 were significantly increased in the epileptic rats following EphB3­Fc injection. The number of DCX­labeled neuronal progenitor cells was reduced in the hippocampus of the epileptic rats. Furthermore, the intensity and frequency of spontaneous recurrent seizures and electroencephalographic seizures were attenuated in the epileptic rats post­injection. These results demonstrated the critical role of ephrin­B3 in regulation of the reelin pathway and hippocampal neurogenesis in epilepsy, providing experimental evidence that ephrin­B3 functions as a potential protective factor in epilepsy, at least in animals.

EphB3 signaling induces cortical endothelial cell death and disrupts the blood-brain barrier after traumatic brain injury.

Damage to the cerebrovascular network is a major contributor to dysfunction in patients suffering from traumatic brain injury (TBI). Vessels are composed of lumen-forming endothelial cells that associate closely with both glial and neuronal units to establish a functional blood-brain barrier (BBB). Under normal physiological conditions, these vascular units play important roles in central nervous system (CNS) homeostasis by delivering oxygen and nutrients while filtering out molecules and cells that could be harmful; however, after TBI this system is disrupted. Here, we describe a novel role for a class of receptors, called dependence receptors, in regulating vessel stability and BBB integrity after CCI injury in mice. Specifically, we identified that EphB3 receptors function as a pro-apoptotic dependence receptor in endothelial cells (ECs) that contributes to increased BBB damage after CCI injury. In the absence of EphB3, we observed increased endothelial cell survival, reduced BBB permeability and enhanced interactions of astrocyte-EC membranes. Interestingly, the brain's response to CCI injury is to reduce EphB3 levels and its ligand ephrinB3; however, the degree and timing of those reductions limit the protective response of the CNS. We conclude that EphB3 is a negative regulator of cell survival and BBB integrity that undermine tissue repair, and represents a protective therapeutic target for TBI patients.

Idiosyncratic Mòjiang virus attachment glycoprotein directs a host-cell entry pathway distinct from genetically related henipaviruses.

In 2012, cases of lethal pneumonia among Chinese miners prompted the isolation of a rat-borne henipavirus (HNV), Mòjiang virus (MojV). Although MojV is genetically related to highly pathogenic bat-borne henipaviruses, the absence of a conserved ephrin receptor-binding motif in the MojV attachment glycoprotein (MojV-G) indicates a differing host-cell recognition mechanism. Here we find that MojV-G displays a six-bladed ß-propeller fold bearing limited similarity to known paramyxoviral attachment glycoproteins, in particular at host receptor-binding surfaces. We confirm the inability of MojV-G to interact with known paramyxoviral receptors in vitro, indicating an independence from well-characterized ephrinB2/B3, sialic acid and CD150-mediated entry pathways. Furthermore, we find that MojV-G is antigenically distinct, indicating that MojV would less likely be detected in existing large-scale serological screening studies focused on well-established HNVs. Altogether, these data indicate a unique host-cell entry pathway for this emerging and potentially pathogenic HNV.

Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord.

BACKGROUND: In the vertebrate spinal cord, motor neurons (MN) are generated in stereotypical numbers from a pool of dedicated progenitors (pMN) whose number depends on signals that control their specification but also their proliferation and differentiation rates. Although the initial steps of pMN specification have been extensively studied, how pMN numbers are regulated over time is less well characterized. RESULTS: Here, we show that ephrinB2 and ephrinB3 are differentially expressed in progenitor domains in the ventral spinal cord with several Eph receptors more broadly expressed. Genetic loss-of-function analyses show that ephrinB2 and ephrinB3 inversely control pMN numbers and that these changes in progenitor numbers correlate with changes in motor neuron numbers. Detailed phenotypic analyses by immunostaining and genetic interaction studies between ephrinB2 and Shh indicate that changes in pMN numbers in ephrin mutants are due to alteration in progenitor identity at late stages of development. CONCLUSIONS: Altogether our data reveal that Eph:ephrin signaling is required to control progenitor identities in the ventral spinal cord.

Ephrin-B3 supports glioblastoma growth by inhibiting apoptosis induced by the dependence receptor EphA4.

EphA4, an Ephrins tyrosine kinase receptor, behaves as a dependence receptor (DR) by triggering cell apoptosis in the absence of its ligand Ephrin-B3. DRs act as conditional tumor suppressors, engaging cell death based on ligand availability; this mechanism is bypassed by overexpression of DRs ligands in some aggressive cancers. The pair EphA4/Ephrin-B3 favors survival of neuronal progenitors of the brain subventricular zone, an area where glioblastoma multiform (GBM) are thought to originate. Here, we report that Ephrin-B3 is highly expressed in human biopsies and that it inhibits EphA4 pro-apoptotic activity in tumor cells. Angiogenesis is directly correlated with GBM aggressiveness and we demonstrate that Ephrin-B3 also supports the survival of endothelial cells in vitro and in vivo. Lastly, silencing of Ephrin-B3 decreases tumor vascularization and growth in a xenograft mice model. Interference with EphA4/Ephrin-B3 interaction could then be envisaged as a relevant strategy to slow GBM growth by enhancing EphA4-induced cell death.

The role of GRIP1 and ephrin B3 in blood pressure control and vascular smooth muscle cell contractility.

Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.

EphrinB1/EphB3b Coordinate Bidirectional Epithelial-Mesenchymal Interactions Controlling Liver Morphogenesis and Laterality.

Positioning organs in the body often requires the movement of multiple tissues, yet the molecular and cellular mechanisms coordinating such movements are largely unknown. Here, we show that bidirectional signaling between EphrinB1 and EphB3b coordinates the movements of the hepatic endoderm and adjacent lateral plate mesoderm (LPM), resulting in asymmetric positioning of the zebrafish liver. EphrinB1 in hepatoblasts regulates directional migration and mediates interactions with the LPM, where EphB3b controls polarity and movement of the LPM. EphB3b in the LPM concomitantly repels hepatoblasts to move leftward into the liver bud. Cellular protrusions controlled by Eph/Ephrin signaling mediate hepatoblast motility and long-distance cell-cell contacts with the LPM beyond immediate tissue interfaces. Mechanistically, intracellular EphrinB1 domains mediate EphB3b-independent hepatoblast extension formation, while EpB3b interactions cause their destabilization. We propose that bidirectional short- and long-distance cell interactions between epithelial and mesenchyme-like tissues coordinate liver bud formation and laterality via cell repulsion.

EphrinB3 restricts endogenous neural stem cell migration after traumatic brain injury.

Traumatic brain injury (TBI) leads to a series of pathological events that can have profound influences on motor, sensory and cognitive functions. Conversely, TBI can also stimulate neural stem/progenitor cell proliferation leading to increased numbers of neuroblasts migrating outside their restrictive neurogenic zone to areas of damage in support of tissue integrity. Unfortunately, the factors that regulate migration are poorly understood. Here, we examine whether ephrinB3 functions to restrict neuroblasts from migrating outside the subventricular zone (SVZ) and rostral migratory stream (RMS). We have previously shown that ephrinB3 is expressed in tissues surrounding these regions, including the overlying corpus callosum (CC), and is reduced after controlled cortical impact (CCI) injury. Our current study takes advantage of ephrinB3 knockout mice to examine the influences of ephrinB3 on neuroblast migration into CC and cortex tissues after CCI injury. Both injury and/or ephrinB3 deficiency led to increased neuroblast numbers and enhanced migration outside the SVZ/RMS zones. Application of soluble ephrinB3-Fc molecules reduced neuroblast migration into the CC after injury and limited neuroblast chain migration in cultured SVZ explants. Our findings suggest that ephrinB3 expression in tissues surrounding neurogenic regions functions to restrict neuroblast migration outside the RMS by limiting chain migration.

Ephrin B3 interacts with multiple EphA receptors and drives migration and invasion in non-small cell lung cancer.

Ephrin receptors (Ephs) are reported to control metastatic signaling of non-small cell lung cancer (NSCLC) and other tumors. Here we show for the first time that blocking expression of the Eph ligand Ephrin B3 inhibits NSCLC cell migration and invasion. We demonstrate that Ephrin B3 directly binds the EphAs EphA2, EphA3, EphA4, and EphA5. EphA2 Ser897 was previously shown to drive migration propensity of tumor cells and our study reveals that EphA2 stays phosphorylated on Ser897 in the Ephrin B3/EphA2 complex in NSCLC cells of different histology. Moreover, we report that within such Ephrin B3/EphA2 complex both Akt Ser 129 and p38MAPK are found indicating a potential to drive migration/proliferation. We also found the EMT marker E-cadherin expression to be maintained or increased upon Ephrin B3 blockade in NSCLC cells. Expression of Ephrin B3 was furthermore analyzed in a cohort of NSCLC stage IA-IB cases (n=200) alongside EphA2 and Ephrin A1. We found that Ephrin B3 was concomitantly expressed with EphA2 and Ephrin A1 with higher Ephrin B3 levels found in non-squamous than in squamous tumors, whereas EphA2 was higher expressed in well-differentiated than in low-differentiated tumors. In the entire NSCLC cohort, Ephrin B3 expression was not linked to patient survival, whereas a high EphA2 expression was associated with improved survival (p=0.03). In conclusion, we show that blocking Ephrin B3 expression inhibits NSCLC proliferation-, migration- and invasion capacity which calls for further studies on interference with Ephrin B3 as a possible therapeutic avenue in this tumor malignancy.