The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin.

Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting.

The peripheral nervous system has astonishing regenerative capabilities in that cut nerves are able to reconnect and re-establish their function. Schwann cells are important players in this process, during which they dedifferentiate to a progenitor/stem cell and promote axonal regrowth. Here, we report that fibroblasts also play a key role. Upon nerve cut, ephrin-B/EphB2 signaling between fibroblasts and Schwann cells results in cell sorting, followed by directional collective cell migration of Schwann cells out of the nerve stumps to guide regrowing axons across the wound. Mechanistically, we find that cell-sorting downstream of EphB2 is mediated by the stemness factor Sox2 through N-cadherin relocalization to Schwann cell-cell contacts. In vivo, loss of EphB2 signaling impaired organized migration of Schwann cells, resulting in misdirected axonal regrowth. Our results identify a link between Ephs and Sox proteins, providing a mechanism by which progenitor cells can translate environmental cues to orchestrate the formation of new tissue.

EphB2 activation in neural stem cells in the basolateral amygdala facilitates neurogenesis and enhances long-term memory.

Many brain diseases lead to a reduction in the number of functional neurons and it would be of value to be able to increase the number of neurons in the affected brain areas. In this study, we examined whether we can promote neural stem cells to produce mature neurons and whether an increase in the mature neurons can affect cognitive performance. We detected that the EphB2 receptor is localized in immature basolateral amygdala (BLA) neurons. We therefore aimed to increase the level of EphB2 activity in neural stem cells (NSCs) in the BLA and examine the effects on the production of mature neurons and cognition. Toward that end, we utilized a photoactivatable EphB2 construct (optoEphB2) to increase EphB2 forward signaling in NSCs in the BLA. We revealed that the activation of optoEphB2 in NSCs in the BLA increased the level of immature and mature neurons in the BLA. We further found that activation of optoEphB2 in BLA NSCs enhanced auditory, but not contextual, long-term fear memory formation. Impairing EphB2 forward signaling did not affect the level of immature and mature neurons in the BLA. This study provides evidence that NSCs can be promoted to produce mature neurons by activating EphB2 to enhance specific brain functions.

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.

Cutting Edge: EPHB2 Is a Coreceptor for Fungal Recognition and Phosphorylation of Syk in the Dectin-1 Signaling Pathway.

Invasive fungal infections have become a leading cause of death among immunocompromised patients, leading to around 1.5 million deaths per year globally. The molecular mechanisms by which hosts defend themselves against fungal infection remain largely unclear, which impedes the development of antifungal drugs and other treatment options. In this article, we show that the tyrosine kinase receptor EPH receptor B2 (EPHB2), together with dectin-1, recognizes ß-glucan and activates downstream signaling pathways. Mechanistically, we found that EPHB2 is a kinase for Syk and is required for Syk phosphorylation and activation after dectin-1 ligand stimulation, whereas dectin-1 is critical for the recruitment of Syk. Ephb2-deficient mice are susceptible to Candida albicans-induced fungemia model, which also supports the role of EPHB2 in antifungal immunity. Overall, we provide evidence that EPHB2 is a coreceptor for the recognition of dectin-1 ligands and plays an essential role in antifungal immunity by phosphorylating Syk.

The ephrin receptor EphB2 regulates the connectivity and activity of enteric neurons.

Highly organized circuits of enteric neurons are required for the regulation of gastrointestinal functions, such as peristaltism or migrating motor complex. However, the factors and molecular mechanisms that regulate the connectivity of enteric neurons and their assembly into functional neuronal networks are largely unknown. A better understanding of the mechanisms by which neurotrophic factors regulate this enteric neuron circuitry is paramount to understanding enteric nervous system (ENS) physiology. EphB2, a receptor tyrosine kinase, is essential for neuronal connectivity and plasticity in the brain, but so far its presence and function in the ENS remain largely unexplored. Here we report that EphB2 is expressed preferentially by enteric neurons relative to glial cells throughout the gut in rats. We show that in primary enteric neurons, activation of EphB2 by its natural ligand ephrinB2 engages ERK signaling pathways. Long-term activation with ephrinB2 decreases EphB2 expression and reduces molecular and functional connectivity in enteric neurons without affecting neuronal density, ganglionic fiber bundles, or overall neuronal morphology. This is highlighted by a loss of neuronal plasticity markers such as synapsin I, PSD95, and synaptophysin, and a decrease of spontaneous miniature synaptic currents. Together, these data identify a critical role for EphB2 in the ENS and reveal a unique EphB2-mediated molecular program of synapse regulation in enteric neurons.

The pathological involvement of spinal cord EphB2 in visceral sensitization in male rats.

Patients with post-traumatic stress disorder (PTSD) are usually at an increased risk for chronic disorders, such as irritable bowel syndrome (IBS), characterized by hyperalgesia and allodynia, but its subsequent effect on visceral hyperalgesia and the mechanism remain unclear. The present study employed single prolonged stress (SPS), a model of PTSD-pain comorbidity, behavioral evaluation, intrathecal drug delivery, immunohistochemistry, Western blotting, and RT-PCR techniques. When detecting visceral sensitivity, the score of the abdominal withdrawal reflex (AWR) induced by graded colorectal distention (CRD) was used. The AWR score was reduced in the SPS day 1 group but increased in the SPS day 7 and SPS day 14 groups at 40 mmHg and 60 mmHg, and the score was increased significantly with EphrinB1-Fc administration. The EphB2+ cell density and EphB2 protein and mRNA levels were downregulated in the SPS day 1 group and then upregulated significantly in the SPS day 7 group; these changes were more noticeable with EphrinB1-Fc administration compared with the SPS-only group. The C-Fos-positive reaction induced by SPS was mainly localized in neurons of the spinal dorsal horn, in which the C-Fos-positive cell density and its protein and mRNA levels were upregulated on SPS days 7 and 14; these changes were statistically significant in the SPS + EphrinB1-Fc group compared with the SPS alone group. The present study confirmed the time window for the AWR value, EphB2 and C-Fos changes, and the effect of EphrinB1-Fc on these changes, which suggests that spinal cord EphB2 activation exacerbates visceral pain after SPS.

The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2.

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.

Loss of ephrin B2 receptor (EPHB2) sets lipid rheostat by regulating proteins DGAT1 and ATGL inducing lipid droplet storage in prostate cancer cells.

Lipid droplet (LD) accumulation in cancer results from aberrant metabolic reprograming due to increased lipid uptake, diminished lipolysis and/or de novo lipid synthesis. Initially implicated in storage and lipid trafficking in adipocytes, LDs are more recently recognized to fuel key functions associated with carcinogenesis and progression of several cancers, including prostate cancer (PCa). However, the mechanisms controlling LD accumulation in cancer are largely unknown. EPHB2, a tyrosine kinase (TKR) ephrin receptor has been proposed to have tumor suppressor functions in PCa, although the mechanisms responsible for these effects are unclear. Given that dysregulation in TRK signaling can result in glutaminolysis we postulated that EPHB2 might have potential effects on lipid metabolism. Knockdown strategies for EPHB2 were performed in prostate cancer cells to analyze the impact on the net lipid balance, proliferation, triacylglycerol-regulating proteins, effect on LD biogenesis, and intracellular localization of LDs. We found that EPHB2 protein expression in a panel of human-derived prostate cancer cell lines was inversely associated with in vivo cell aggressiveness. EPHB2 silencing increased the proliferation of prostate cancer cells and concurrently induced de novo LD accumulation in both cytoplasmic and nuclear compartments as well as a "shift" on LD size distribution in newly formed lipid-rich organelles. Lipid challenge using oleic acid exacerbated the effects on the LD phenotype. Loss of EPHB2 directly regulated key proteins involved in maintaining lipid homeostasis including, increasing lipogenic DGAT1, DGAT2 and PLIN2 and decreasing lipolytic ATGL and PEDF. A DGAT1-specific inhibitor abrogated LD accumulation and proliferative effects induced by EPHB2 loss. In conclusion, we highlight a new anti-tumor function of EPHB2 in lipid metabolism through regulation of DGAT1 and ATGL in prostate cancer. Blockade of DGAT1 in EPHB2-deficient tumors appears to be effective in restoring the lipid balance and reducing tumor growth.

EphB2 activates CREB-dependent expression of Annexin A1 to regulate dendritic spine morphogenesis.

Dendritic spines are the postsynaptic structure to mediate signal transduction in neural circuitry, whose function and plasticity are regulated by organization of their molecular architecture and by the expression of target genes and proteins. EphB2, a member of the Eph receptor tyrosine kinase family, potentiates dendritic spine maturation through cytoskeleton reorganization and protein trafficking. However, the transcriptional mechanisms underlying prolonged activation of EphB2 signaling during dendritic spine morphogenesis are unknown. Herein, we performed transcriptional profiling by stimulating EphB2 signaling and identified differentially expressed genes implicated in pivotal roles at synapses. Notably, we characterized an F-actin binding protein, Annexin A1, whose expression was induced by EphB2 signaling; the promotor activity of its coding gene Anxa1 is regulated by the activity of CREB (cAMP-response element-binding protein). Knockdown of Annexin A1 led to a significant reduction of mature dendritic spines without an obvious deficit in the complexity of dendrites. Altogether, our findings suggest that EphB2-induced, CREB-dependent Annexin A1 expression plays a key role in regulating dendritic spine morphology.

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.

The Ephb2 Receptor Uses Homotypic, Head-to-Tail Interactions within Its Ectodomain as an Autoinhibitory Control Mechanism.

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell-cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph-ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph-Eph interfaces. One Eph-Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor-receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.

The Value of EphB2 Receptor and Cognate Ephrin Ligands in Prognostic and Predictive Assessments of Human Breast Cancer.

Cell-cell communication proteins Eph and ephrin constitute the largest family of receptor tyrosine kinases (RTKs). They are distinguished by the fact that both receptors and ligands are membrane-bound, and both can drive intracellular signaling in their respective cells. Ever since these RTKs have been found to be involved in cancer development, strategies to target them therapeutically have been actively pursued. However, before this goal can be rationally achieved, the contributions of either Eph receptors or their ephrin ligands to cancer development and progression should be scrutinized in depth. To assess the clinical pertinence of this concern, we performed a systematic review and meta-analysis of the prognostic/predictive value of EphB2 and its multiple cognate ephrin ligands in breast cancer. We found that EphB2 has prognostic value, as indicated by the association of higher EphB2 expression levels with lower distant metastasis-free survival (DMFS), and the association of lower EphB2 expression levels with poorer relapse-free survival (RFS). We also found that higher EphB2 expression could be a prognostic factor for distant metastasis, specifically in the luminal subtypes of breast cancer. EFNB2 showed a marked correlation between higher expression levels and shorter DMFS. EFNA5 or EFNB1 overexpression is correlated with longer RFS. Increased EFNB1 expression is correlated with longer OS in lymph node (LN)-negative patients and the luminal B subtype. Higher levels of EFNB2 or EFNA5 are significantly correlated with shorter RFS, regardless of LN status. However, while this correlation with shorter RFS is true for EFNB2 in all subtypes except basal, it is also true for EFNA5 in all subtypes except HER2+. The analysis also points to possible predictive value for EphB2. In systemically treated patients who have undergone either endocrine therapy or chemotherapy, we found that higher expression of EphB2 is correlated with better rates of RFS. Bearing in mind the limitations inherent to any mRNA-based profiling method, we complemented our analysis with an immunohistochemical assessment of expression levels of both the EphB2 receptor and cognate ephrin ligands. We found that the latter are significantly more expressed in cancers than in normal tissues, and even more so in invasive and metastatic samples than in ductal carcinoma in situ (DCIS). Finally, in an in vitro cellular model of breast cancer progression, based on H-Ras-transformation of the MCF10A benign mammary cell line, we observed dramatic increases in the mRNA expression of EphB2 receptor and EFNB1 and EFNB2 ligands in transformed and invasive cells in comparison with their benign counterparts. Taken together, these data show the clinical validity of a model whereby EphB2, along with its cognate ephrin ligands, have dual anti- and pro-tumor progression effects. In so doing, they reinforce the necessity of further biological investigations into Ephs and ephrins, prior to using them in targeted therapies.

Eps15R and clathrin regulate EphB2-mediated cell repulsion.

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans-endocytosis of the inter-cellular receptor-ligand EphB-ephrinB complex is required. The molecular mechanism underlying trans-endocytosis is poorly defined. Here we show that the process is clathrin- and Eps15R-mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co-cultures of EphB2- and ephrinB1-expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2-mediated cell repulsion as shown in a rescue experiment in the EphB2 co-culture assay where wild type Eps15R but not the clathrin-binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans-endocytosis and thereby cell repulsion.

Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

A mutation of the human EPHB2 gene leads to a major platelet functional defect.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbß3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbß3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.

Patients with irritable bowel syndrome (IBS) show pain hypersensitivity and smooth muscle hypercontractility in response to colorectal distension (CRD). Synaptic plasticity, a key process of memory formation, in the enteric nervous system may be a novel explanation. This study aimed to explore the regulatory role of ephrinB2/ephB2 in enteric synaptic plasticity and colonic hyperreactive motility in IBS. Postinfectious (PI)-IBS was induced by Trichinella spiralis infection in rats. Isometric contractions of colonic circular muscle strips, particularly neural-mediated contractions, were recorded ex vivo. Meanwhile, ephrinB2/ephB2-mediated enteric structural and functional synaptic plasticity were assessed in the colonic muscularis, indicating that ephrinB2 and ephB2 were located on enteric nerves and up-regulated in the colonic muscularis of PI-IBS rats. Colonic hypersensitivity to CRD and neural-mediated colonic hypercontractility were present in PI-IBS rats, which were correlated with increased levels of cellular homologous fos protein (c-fos) and activity-regulated cystoskeleton-associated protein (arc), the synaptic plasticity-related immediate early genes, and were ameliorated by ephB2Fc (an ephB2 receptor blocker) or MK801 (an NMDA receptor inhibitor) exposure. EphrinB2/ephB2 facilitated synaptic sprouting and NMDA receptor-mediated synaptic potentiation in the colonic muscularis of PI-IBS rats and in the longitudinal muscle-myenteric plexus cultures, involving the Erk-MAPK and PI3K-protein kinase B pathways. In conclusion, ephrinB2/ephB2 promoted the synaptic sprouting and potentiation of myenteric nerves involved in persistent muscle hypercontractility and pain in PI-IBS. Hence, ephrinB2/ephB2 may be an emerging target for the treatment of IBS.-Zhang, L., Wang, R., Bai, T., Xiang, X., Qian, W., Song, J., Hou, X. EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.

Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.

Association between rs12742784 polymorphism and hip fracture, bone mineral density, and EPHB2 mRNA expression levels in elderly Chinese women.

Objective: This study aimed to determine the association between rs12742784 polymorphism in the non-coding area and hip fracture, bone mineral density (BMD), and EPHB2 mRNA expression levels in elderly Chinese women.Methods: We investigated 250 Chinese women (mean age: 63.5 ± 8.3 years) including 123 hip fracture patients and 127 non-fracture controls. All participants underwent clinical examination to meet the inclusion criteria. Lumbar and hip BMD were detected by dual-energy X-ray absorptiometry. rs12742784 polymorphism was determined by restriction fragment length polymorphism and EPHB2 mRNA expression levels were measured by real-time polymerase chain reaction.Results: Distribution of rs12742784 genotypes agreed with Hardy-Weinberg equilibrium. The frequency of the CT + TT genotype was significantly associated with decreased risk of hip fracture (adjusted odds ratio = 0.57, p < 0.01) after adjusting for age and body mass index, and with increased BMD and EPHB2 mRNA expression levels. The T allele of the rs12742784 single nucleotide polymorphism (SNP) was a protective factor for hip fracture (adjusted odds ratio = 0.56, p < 0.01).Conclusion: rs12742784 polymorphism was associated with EPHB2 mRNA expression levels, BMD, and hip fracture in Chinese women. The T allele of the rs12742784 SNP was a protective factor for osteoporosis and hip fracture.

Syk kinases are required for spinal commissural axon repulsion at the midline via the ephrin/Eph pathway.

In the hematopoietic system, Syk family tyrosine kinases are essential components of immunoreceptor ITAM-based signaling. While there is increasing data indicating the involvement of immunoreceptors in neural functions, the contribution of Syk kinases remains obscure. Previously, we identified phosphorylated forms of Syk kinases in specialized populations of migrating neurons or projecting axons. Moreover, we identified ephrin/Eph as guidance molecules utilizing the ITAM-bearing CD3zeta (Cd247) and associated Syk kinases for the growth cone collapse response induced in vitro Here, we show that in the developing spinal cord, Syk is phosphorylated in navigating commissural axons. By analyzing axon trajectories in open-book preparations of Syk(-/-); Zap70(-/-) mouse embryos, we show that Syk kinases are dispensable for attraction towards the midline but confer growth cone responsiveness to repulsive signals that expel commissural axons from the midline. Known to serve a repulsive function at the midline, ephrin B3/EphB2 are obvious candidates for driving the Syk-dependent repulsive response. Indeed, Syk kinases were found to be required for ephrin B3-induced growth cone collapse in cultured commissural neurons. In fragments of commissural neuron-enriched tissues, Syk is in a constitutively phosphorylated state and ephrin B3 decreased its level of phosphorylation. Direct pharmacological inhibition of Syk kinase activity was sufficient to induce growth cone collapse. In conclusion, Syk kinases act as a molecular switch of growth cone adhesive and repulsive responses.