Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression.

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain.

Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.

Hippocampal Lnx1-NMDAR multiprotein complex mediates initial social memory.

Social interaction and communication are evolutionary conserved behaviours that are developed in mammals to establish partner cognition. Deficit in sociability has been represented in human patients and animal models of neurodevelopmental disorders, which are connected with genetic variants of synaptic glutamate receptors and associated PDZ-binding proteins. However, it remains elusive how these key proteins are specialized in the cellular level for the initial social behaviour during postnatal developmental stage. Here we identify a hippocampal CA3 specifically expressed PDZ scaffold protein Lnx1 required for initial social behaviour. Through gene targeting we find that Lnx1 deficiency led to a hippocampal subregional disorder in neuronal activity and social memory impairments for partner discrimination observed in juvenile mice which also show cognitive defects in adult stage. We further demonstrate that Lnx1 deletion causes NMDA receptor (NMDAR) hypofunction and this is attributable to decreased GluN2B expression in PSD compartment and disruption of the Lnx1-NMDAR-EphB2 complex. Specific restoration of Lnx1 or EphB2 protein in the CA3 area of Lnx1-/- mice rescues the defective synaptic function and social memory. These findings thus reveal crucial roles of postsynaptic NMDAR multiprotein complex that regulates the formation of initial social memory during the adolescent period.

Key role for EphB2 receptor in kidney fibrosis.

Erythropoietin producing hepatocellular (Eph)-Eph receptor interacting (Ephrin) receptor-ligand signaling has been implicated in the development of tissue fibrosis, though it has not been well defined in the kidney. We detected substantial up-regulation of expression and phosphorylation of the EphB2 receptor tyrosine kinase in fibrotic kidney tissue obtained both from mice subjected to the unilateral renal ischemia-reperfusion (IR) model at 14 days and in patients suffering from chronic kidney disease (CKD). Knockout (KO) mice lacking EphB2 expression exhibited a normal renal structure and function, indicating no major role for this receptor in kidney development or action. Although IR injury is well-known to cause tissue damage, fibrosis, and renal dysfunction, we found that kidneys from EphB2KO mice showed much less renal tubular injury and retained a more preserved renal function. IR-injured kidneys from EphB2 KOs exhibited greatly reduced fibrosis and inflammation compared with injured wildtype (WT) littermates, and this correlated with a significant reduction in renal expression of profibrotic molecules, inflammatory cytokines, NADPH oxidases, and markers for cell proliferation, tubular epithelial-to-mesenchymal transition (EMT), myofibroblast activation, and apoptosis. A panel of 760 fibrosis-associated genes were further assessed, revealing that 506 genes in WT mouse kidney following IR injury changed their expression. However, 70.9% of those genes were back to or close to normal in expression when EphB2 was deleted. These data indicate that endogenous EphB2 expression and signaling are abnormally activated after kidney injury and subsequently contribute to the development of renal fibrosis via regulation of multiple profibrotic pathways.

Autonomous and non-autonomous roles for ephrin-B in interneuron migration.

While several studies indicate the importance of ephrin-B/EphB bidirectional signaling in excitatory neurons, potential roles for these molecules in inhibitory neurons are largely unknown. We identify here an autonomous receptor-like role for ephrin-B reverse signaling in the tangential migration of interneurons into the neocortex using ephrin-B (EfnB1/B2/B3) conditional triple mutant (TMlz) mice and a forebrain inhibitory neuron specific Cre driver. Inhibitory neuron deletion of the three EfnB genes leads to reduced interneuron migration, abnormal cortical excitability, and lethal audiogenic seizures. Truncated and intracellular point mutations confirm the importance of ephrin-B reverse signaling in interneuron migration and cortical excitability. A non-autonomous ligand-like role was also identified for ephrin-B2 that is expressed in neocortical radial glial cells and required for proper tangential migration of GAD65-positive interneurons. Our studies thus define both receptor-like and ligand-like roles for the ephrin-B molecules in controlling the migration of interneurons as they populate the neocortex and help establish excitatory/inhibitory (E/I) homeostasis.

Abnormalities in cortical interneuron subtypes in ephrin-B mutant mice.

To explore roles for ephrin-B/EphB signaling in cortical interneurons, we previously generated ephrin-B (Efnb1/b2/b3) conditional triple mutant (TMlz ) mice using a Dlx1/2.Cre inhibitory neuron driver and green fluorescent protein (GFP) reporters for the two main inhibitory interneuron groups distinguished by expression of either glutamic acid decarboxylase 1 (GAD1; GAD67-GFP) or 2 (GAD2; GAD65-GFP). This work showed a general involvement of ephrin-B in migration and population of interneurons into the embryonic neocortex. We now determined whether specific interneurons are selectively affected in the adult brains of TMlz .Cre mice by immunostaining with antibodies that identify the different subtypes. The results indicate that GAD67-GFP-expressing interneurons that also express parvalbumin (PV), calretinin (CR) and, to a lesser extent, somatostatin (SST) and Reelin (Rln) were significantly reduced in the cortex and hippocampal CA1 region in TMlz .Cre mutant mice. Neuropeptide Y (NPY) interneurons that also express GAD67-GFP were reduced in the hippocampal CA1 region, but much less so in the cortex, although these cells exhibited abnormal cortical layering. In GAD65-GFP-expressing interneurons, CR subtypes were reduced in both cortex and hippocampal CA1 region, whereas Rln interneurons were reduced exclusively in hippocampus, and the numbers of NPY and vasoactive intestinal polypeptide (VIP) subtypes appeared normal. PV and CR subtype interneurons in TMlz .Cre mice also exhibited reductions in their perisomatic area, suggesting abnormalities in dendritic/axonal complexity. Altogether, our data indicate that ephrin-B expression within forebrain interneurons is required in specific subtypes for their normal population, cortical layering and elaboration of cell processes.

Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear.

UNLABELLED: The amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to environmental aversive cues. However, the molecular mechanism underlying the initial neuron responses is poorly understood. In this study, we monitored the innate defensive responses to aversive stimuli of either elevated plus maze or predator odor in juvenile mice and found that glutamatergic neurons were activated in amygdala. Loss of EphB2, a receptor tyrosine kinase expressed in amygdala neurons, suppressed the reactions and led to defects in spine morphogenesis and fear behaviors. We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2. A constitutively active form of EphB2 was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3, a brain-enriched ligand to EphB2. These data suggest that kinase-dependent EphB2 intracellular signaling plays a major role for innate fear responses during the critical developing period, in which spinogenesis in amygdala glutamatergic neurons was involved. SIGNIFICANCE STATEMENT: Generation of innate fear responses to threat as an evolutionally conserved brain feature relies on development of functional neural circuit in amygdala, but the molecular mechanism remains largely unknown. We here identify that EphB2 receptor tyrosine kinase, which is specifically expressed in glutamatergic neurons, is required for the innate fear responses in the neonatal brain. We further reveal that EphB2 mediates coordination of spinogenesis and neuron activation in amygdala during the critical period for the innate fear. EphB2 catalytic activity plays a major role for the behavior upon EphB-ephrin-B3 binding and transnucleus neuronal connections. Our work thus indicates an essential synaptic molecular signaling within amygdala that controls synapse development and helps bring about innate fear emotions in the postnatal developing brain.

Eph receptor interclass cooperation is required for the regulation of cell proliferation.

Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells.

EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding.

In early brain development, ascending thalamocortical axons (TCAs) navigate through the ventral telencephalon (VTel) to reach their target regions in the young cerebral cortex. Descending, deep-layer cortical axons subsequently target appropriate thalamic and subcortical target regions. However, precisely how and when corticothalamic axons (CTAs) identify their appropriate, reciprocal thalamic targets remains unclear. We show here that EphB1 and EphB2 receptors control proper navigation of a subset of TCA and CTA projections through the VTel. We show in vivo that EphB receptor forward signaling and the ephrinB1 ligand are required during the early navigation of L1-CAM(+) thalamic fibers in the VTel, and that the misguided thalamic fibers in EphB1/2 KO mice appear to interact with cortical subregion-specific axon populations during reciprocal cortical axon guidance. As such, our findings suggest that descending cortical axons identify specific TCA subpopulations in the dorsal VTel to coordinate reciprocal cortical-thalamic connectivity in the early developing brain.

Molecular mechanisms controlling the migration of striatal interneurons.

In the developing telencephalon, the medial ganglionic eminence (MGE) generates many cortical and virtually all striatal interneurons. While the molecular mechanisms controlling the migration of interneurons to the cortex have been extensively studied, very little is known about the nature of the signals that guide interneurons to the striatum. Here we report that the allocation of MGE-derived interneurons in the developing striatum of the mouse relies on a combination of chemoattractive and chemorepulsive activities. Specifically, interneurons migrate toward the striatum in response to Nrg1/ErbB4 chemoattraction, and avoid migrating into the adjacent cortical territories by a repulsive activity mediated by EphB/ephrinB signaling. Our results also suggest that the responsiveness of MGE-derived striatal interneurons to these cues is at least in part controlled by the postmitotic activity of the transcription factor Nkx2-1. This study therefore reveals parallel mechanisms for the migration of MGE-derived interneurons to the striatum and the cerebral cortex.

Ephrin-B/EphB Signaling Is Required for Normal Innervation of Lingual Gustatory Papillae.

The innervation of taste buds is an excellent model system for studying the guidance of axons during targeting because of their discrete nature and the high fidelity of innervation. The pregustatory epithelium of fungiform papillae is known to secrete diffusible axon guidance cues such as BDNF and Sema3A that attract and repel, respectively, geniculate ganglion axons during targeting, but diffusible factors alone are unlikely to explain how taste axon terminals are restricted to their territories within the taste bud. Nondiffusible cell surface proteins such as Ephs and ephrins can act as receptors and/or ligands for one another and are known to control axon terminal positioning in several parts of the nervous system, but they have not been studied in the gustatory system. We report that ephrin-B2 linked ß-galactosidase staining and immunostaining was present along the dorsal epithelium of the mouse tongue as early as embryonic day 15.5 (E15.5), but was not detected at E14.5, when axons first enter the epithelium. Ephrin-B1 immunolabeling was barely detected in the epithelium and found at a somewhat higher concentration in the mesenchyme subjacent to the epithelium. EphB1 and EphB2 were detected in lingual sensory afferents in vivo and geniculate neurites in vitro. Ephrin-B1 and ephrin-B2 were similarly effective in repelling or suppressing outgrowth by geniculate neurites in vitro. These in vitro effects were independent of the neurotrophin used to promote outgrowth, but were reduced by elevated levels of laminin. In vivo, mice null for EphB1 and EphB2 exhibited decreased gustatory innervation of fungiform papillae. These data provide evidence that ephrin-B forward signaling is necessary for normal gustatory innervation of the mammalian tongue.

Ephrin-B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear.

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB-Efnb2 signaling-deficient mice.

Myelin-derived ephrinB3 restricts axonal regeneration and recovery after adult CNS injury.

Recovery of neurological function after traumatic injury of the adult mammalian central nervous system is limited by lack of axonal growth. Myelin-derived inhibitors contribute to axonal growth restriction, with ephrinB3 being a developmentally important axonal guidance cue whose expression in mature oligodendrocytes suggests a role in regeneration. Here we explored the in vivo regeneration role of ephrinB3 using mice lacking a functional ephrinB3 gene. We confirm that ephrinB3 accounts for a substantial portion of detergent-resistant myelin-derived inhibition in vitro. To assess in vivo regeneration, we crushed the optic nerve and examined retinal ganglion fibers extending past the crush site. Significantly increased axonal regeneration is detected in ephrinB3(-/-) mice. Studies of spinal cord injury in ephrinB3(-/-) mice must take into account altered spinal cord development and an abnormal hopping gait before injury. In a near-total thoracic transection model, ephrinB3(-/-) mice show greater spasticity than wild-type mice for 2 mo, with slightly greater hindlimb function at later time points, but no evidence for axonal regeneration. After a dorsal hemisection injury, increased corticospinal and raphespinal growth in the caudal spinal cord are detected by 6 wk. This increased axonal growth is accompanied by improved locomotor performance measured in the open field and by kinematic analysis. Thus, ephrinB3 contributes to myelin-derived axonal growth inhibition and limits recovery from adult CNS trauma.

Ephrin reverse signaling in axon guidance and synaptogenesis.

Axon-cell and axon-dendrite contact is a highly regulated process necessary for the formation of precise neural circuits and a functional neural network. Eph-ephrin interacting molecules on the membranes of axon nerve terminals and target dendrites act as bidirectional ligands/receptors to transduce signals into both the Eph-expressing and ephrin-expressing cells to regulate cytoskeletal dynamics. In particular, recent evidence indicates that ephrin reverse signal transduction events are important in controlling both axonal and dendritic elaborations of neurons in the developing nervous system. Here we review how ephrin reverse signals are transduced into neurons to control maturation of axonal pre-synaptic and dendritic post-synaptic structures.

EphB2 receptor forward signaling controls cortical growth cone collapse via Nck and Pak.

EphB receptors and their ephrinB ligands transduce bidirectional signals that mediate contact-dependent axon guidance primarily by promoting growth cone repulsion. However, how EphB receptor-mediated forward signaling induces axonal repulsion remains poorly understood. Here, we identify Nck and Pak proteins as essential forward signaling components of EphB2-dependent growth cone collapse in cortical neurons. We show that kinase-active EphB2 binds to Pak and promotes growth cone repulsion via Pak kinase activity, Pak-Nck binding, RhoA signaling and endocytosis. However, Pak's function in this context appears to be independent of Rac/Cdc42-GTP, consistent with the absence of Rac-GTP production after ephrinB treatment of cortical neurons. Taken together, our findings suggest that ephrinB-activated EphB2 receptors recruit a novel Nck/Pak signaling complex to mediate repulsive cortical growth cone guidance, which may be relevant for EphB forward signaling-dependent axon guidance in vivo.

EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis.

OBJECTIVE: Erythropoietin-producing hepatocellular (Eph)/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). METHODS: We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models of skin fibrosis. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from patients with SSc and healthy controls, which was followed by in vivo analysis of fibroblast-specific Ephb2-deficient mice. RESULTS: Expression of EphB2 is up-regulated in SSc skin tissue and explanted SSc dermal fibroblasts compared with healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-ß (TGF-ß) cytokines up-regulate EphB2 in dermal fibroblasts via noncanonical TGF-ß/mother against decapentaplegic signaling, and silencing EPHB2 in human dermal fibroblasts is sufficient to dampen TGF-ß-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. CONCLUSION: Our data implicate TGF-ß regulation of EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

Ephrin-B2 reverse signaling increases α5ß1 integrin-mediated fibronectin deposition and reduces distal lung compliance.

Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of ephrin-B2 reverse signaling in the lung was assessed in Efnb2(LacZ/6YFΔV) mutants that coexpress the intracellular truncated ephrin-B2-ß-galactosidase fusion and an intracellular point mutant ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain-containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the α5 integrin receptor required for FN fibrillogenesis. These experiments indicate that ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.

A novel feedback mechanism by Ephrin-B1/B2 in T-cell activation involves a concentration-dependent switch from costimulation to inhibition.

Bidirectional signals via Eph receptors/ephrins have been recognized as major forms of contact-dependent cell communications such as cell attraction and repulsion. T cells express EphBs, and their ligands, the ephrin-Bs, have been known as costimulatory molecules for T-cell proliferation. Recently, another remarkable feature of ephrin-As has emerged in the form of a concentration-dependent transition from promotion to inhibition in axon growth. Here we examined whether this modification plays a role in ephrin-B costimulation in murine primary T cells. Low doses of ephrin-B1 and ephrin-B2 costimulated T-cell proliferation induced by anti-CD3, but high concentrations strongly inhibited it. In contrast, ephrin-B3 showed a steadily increasing stimulatory effect. This modulation was virtually preserved in T cells from mice simultaneously lacking four genes, EphB1, EphB2, EphB3, and EphB6. High concentrations of ephrin-B1/B2, but not ephrin-B3, inhibited the anti-CD3-induced phosphorylation of Lck and its downstream signals such as Erk and Akt. Additionally, high doses of any ephrin-Bs could phosphorylate EphB4. However, only ephrin-B1/B2 but not ephrin-B3 recruited SHP1, a phosphatase to suppress the phosphorylation of Lck. These data suggest that EphB4 signaling could engage in negative feedback to TCR signals. T-cell activation may be finely adjusted by the combination and concentration of ephrin-Bs expressed in the immunological microenvironment.

Epithelial dynamics of pancreatic branching morphogenesis.

The mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching, however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.