Silencing EPHB2 diminished the malignant biological properties of esophagus cancer cells by blocking autophagy and Wnt/ß-catenin pathway.

Eph receptor B2 (EPHB2) is overexpressed in some tumors and relevant to unfavorable outcomes of tumor patients. By searching Gene Expression Profiling Interactive Analysis and KM Plot websites, we discovered that EPHB2 was highly expressed in patients with esophageal cancer, leading to poor prognosis. However, the role and molecular mechanism of EPHB2 in esophagus cancer is unknown. Our study aims to unveil the underlying mechanism by which EPHB2 modulates the biological properties of esophagus cancer cells. After si-EPHB2 transfection, the malignant biological properties of esophagus cancer cells were determined by several biological experiments. IWP-4 was applied to block Wnt/ß-catenin signaling pathway. The expressions of autophagy and Wnt/ß-catenin signaling pathway relevant molecules were tested by western blot assay. An increased expression of EPHB2 was happened in esophagus cancer samples and loss of EPHB2 diminished esophagus cancer cells proliferation, migration, and invasion. Moreover, our data showed that depletion of EPHB2 blocked the autophagy and in-activated Wnt/ß-catenin signaling pathway in esophagus cancer cells. While, IWP-4 treatment inhibited the autophagy and limited esophagus cancer cells proliferation, migration, and invasion. Moreover, EPHB2 knocked down strengthened the effect of IWP-4 treatment in regulating esophagus cancer cells proliferation, migration, and invasion. Finally, we illustrated that EPHB2 regulated the biological properties of esophagus cancer cells by modulating autophagy and Wnt/ß-catenin signaling pathway. Our study illustrated that EPHB2 might be a worthwhile target considering for the treatment of esophagus cancer.

Cysteine sulfenylation contributes to liver fibrosis via the regulation of EphB2-mediated signaling.

Sulfenylation is a reversible oxidative posttranslational modification (PTM) of proteins on cysteine residues. Despite the dissection of various biological functions of cysteine sulfenylation, its roles in hepatic fibrosis remain elusive. Here, we report that EphB2, a receptor tyrosine kinase previously implicated in liver fibrosis, is regulated by cysteine sulfenylation during the fibrotic progression of liver. Specifically, EphB2 is sulfenylated at the residues of Cys636 and Cys862 in activated hepatic stellate cells (HSCs), leading to the elevation of tyrosine kinase activity and protein stability of EphB2 and stronger interactions with focal adhesion kinase for the activation of downstream mitogen-activated protein kinase signaling. The inhibitions of both EphB2 kinase activity and cysteine sulfenylation by idebenone (IDE), a marketed drug with potent antioxidant activity, can markedly suppress the activation of HSCs and ameliorate hepatic injury in two well-recognized mouse models of liver fibrosis. Collectively, this study reveals cysteine sulfenylation as a new type of PTM for EphB2 and sheds a light on the therapeutic potential of IDE for the treatment of liver fibrosis.

Comprehensive pan-cancer analysis reveals EPHB2 is a novel predictive biomarker for prognosis and immunotherapy response.

PURPOSE: Recent studies have increasingly linked Ephrin receptor B2 (EPHB2) to cancer progression. However, comprehensive investigations into the immunological roles and prognostic significance of EPHB2 across various cancers remain lacking. METHODS: We employed various databases and bioinformatics tools to investigate the impact of EPHB2 on prognosis, immune infiltration, genome instability, and response to immunotherapy. Validation of the correlation between EPHB2 expression and M2 macrophages included analyses using bulk and single-cell transcriptomic datasets, spatial transcriptomics, and multi-fluorescence staining. Moreover, we performed cMap web tool to screen for EPHB2-targeted compounds and assessed their potential through molecular docking and dynamics simulations. Additionally, in vitro validation using lung adenocarcinoma (LUAD) cell lines was conducted to confirm the bioinformatics predictions about EPHB2. RESULTS: EPHB2 dysregulation was observed across multiple cancer types, where it demonstrated significant diagnostic and prognostic value. Gene Set Enrichment Analysis (GSEA) indicated that EPHB2 is involved in enhancing cellular proliferation, invasiveness of cancer cells, and modulation of the anti-cancer immune response. Furthermore, it is emerged as a pan-cancer marker for M2 macrophage infiltration, supported by integrated analyses of transcriptomics and multiple fluorescence staining. In LUAD cells, knockdown of EPHB2 expression led to a decrease in both cell proliferation and migratory activity. CONCLUSION: EPHB2 expression may serve as a pivotal indicator of M2 macrophage infiltration, offering vital insights into tumor dynamics and progression across various cancers, including lung adenocarcinoma, highlighting its significant prognostic and therapeutic potential for further exploration.

ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans.

Ephrin-B-EphB signaling can promote pain through ligand-receptor interactions between peripheral cells, like immune cells expressing ephrin-Bs, and EphB receptors expressed by DRG neurons. Previous studies have shown increased ephrin-B2 expression in peripheral tissues like synovium of rheumatoid and osteoarthritis patients, indicating the clinical significance of this signaling. The primary goal of this study was to understand how ephrin-B2 acts on mouse and human DRG neurons, which express EphB receptors, to promote pain and nociceptor plasticity. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2 injection either to the paw or the cranial dura. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by eFT508, a specific MNK inhibitor. Sensory neuron-specific knockout of EphB2 using Pirt-Cre demonstrated that ephrin-B2 actions require this receptor. In Ca2+-imaging experiments on cultured DRG neurons, ephrin-B2 treatment enhanced Ca2+ transients in response to PGE2 and these effects were absent in DRG neurons from MNK1-/- and EphB2-PirtCre mice. In experiments on human DRG neurons, ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+ responses to PGE2 treatment, both blocked by eFT508. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling, offering new insight into how ephrin-B signaling promotes pain.

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.

Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release.

At the synapse, presynaptic neurotransmitter release is tightly controlled by release machinery, involving the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and Munc13. The Ca2+ sensor Doc2 cooperates with Munc13 to regulate neurotransmitter release, but the underlying mechanisms remain unclear. In our study, we have characterized the binding mode between Doc2 and Munc13 and found that Doc2 originally occludes Munc13 to inhibit SNARE complex assembly. Moreover, our investigation unveiled that EphB2, a presynaptic adhesion molecule (SAM) with inherent tyrosine kinase functionality, exhibits the capacity to phosphorylate Doc2. This phosphorylation attenuates Doc2 block on Munc13 to promote SNARE complex assembly, which functionally induces spontaneous release and synaptic augmentation. Consistently, application of a Doc2 peptide that interrupts Doc2-Munc13 interplay impairs excitatory synaptic transmission and leads to dysfunction in spatial learning and memory. These data provide evidence that SAMs modulate neurotransmitter release by controlling SNARE complex assembly.

The lack of EphB3 receptor prevents bone loss in mouse models of osteoporosis.

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment.

Osteoporosis affects more than 200 million people, mostly women. Our work shows that the EphB3 receptor restricts bone formation, and its absence prevents bone loss in osteoporotic mice. The bone protection observed in EphB3-deficient mice is due to the presence of more bone-forming cells and fewer bone-degrading cells. Molecularly, we found that when there's no EphB3 in mesenchymal stem cells, some bone-promoting genes are increased while many inhibitors are reduced. Therefore, this receptor could become a key target for new therapies that would help to improve the quality of life for those suffering from bone diseases. We're really excited to share our findings with a broad audience, including patients, healthcare professionals, researchers, and the life sciences industry.

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.

Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in Caenorhabditis elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.

Interaction between the EPHB2 receptor and EFNB1 ligand drives gastric cancer invasion and metastasis via the Wnt/ß-catenin/FAK pathway.

The survival benefit for patients with gastric cancer (GC) is modest due to its high transfer potential. Targeted therapy for metastasis-related genes in GC may be a viable approach, however, inhibitors specifically targeting GC are limited. In this study, GC patient-derived xenografts (PDX) with metastatic burden were established via orthotopic transplantation. PCR-Array analysis of primary and metastatic tumors revealed EPH receptor B2 (EPHB2) as the most significantly upregulated gene. The interaction between the EPHB2 receptor and its cognate-specific EFNB1 ligands was high in GC and correlated with a poor prognosis. Fc-EFNB1 treatment increased the invasion and migration abilities of GC cells and induced a high EPHB2 expression. EPHB2 knockdown in GC cells completely abolished the ephrin ligand-induced effects on invasion and migration abilities. Signal transduction analysis revealed Wnt/ß-catenin and FAK as downstream signaling mediators potentially inducing the EPHB2 phenotype. In conclusion, the observed deregulation of EPHB2/EFNB1 expression in GC enhances the invasive phenotype, suggesting a potential role of EPHB2/EFNB1 compound in local tumor cell invasion and the formation of metastasis.

Discovery and Characterization of Ephrin B2 and EphB4 Dysregulation and Novel Mutations in Cerebral Cavernous Malformations: In Vitro and Patient-Derived Evidence of Ephrin-Mediated Endothelial Cell Pathophysiology.

Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.

EphB2-dependent prefrontal cortex activation promotes long-range social approach and partner responsiveness.

Social behavior starts with dynamic approach prior to the final consummation. The flexible processes ensure mutual feedback across social brains to transmit signals. However, how the brain responds to the initial social stimuli precisely to elicit timed behaviors remains elusive. Here, by using real-time calcium recording, we identify the abnormalities of EphB2 mutant with autism-associated Q858X mutation in processing long-range approach and accurate activity of prefrontal cortex (dmPFC). The EphB2-dependent dmPFC activation precedes the behavioral onset and is actively associated with subsequent social action with the partner. Furthermore, we find that partner dmPFC activity is responsive coordinately to the approaching WT mouse rather than Q858X mutant mouse, and the social defects caused by the mutation are rescued by synchro-optogenetic activation in dmPFC of paired social partners. These results thus reveal that EphB2 sustains neuronal activation in the dmPFC that is essential for the proactive modulation of social approach to initial social interaction.

Fear Conditioning Leads to Enduring Alterations in RNA Transcripts in Hippocampal Neuropil that are Dependent on EphB2 Forward Signaling.

Alterations in mRNA transcription have been associated with changes in brain functions. We wanted to examine if fear conditioning causes long-term changes in transcriptome profiles in the basolateral amygdala (BLA) and hippocampus using RNA-Seq and laser microdissection microscopy. We further aimed to uncover whether these changes are involved in memory formation by monitoring their levels in EphB2lacZ/lacZ mice, which lack EphB2 forward signaling and can form short-term fear conditioning memory but not long-term fear conditioning memory. We found transcriptome signatures unique to each brain region that are comprise of specific cellular pathways. We also revealed that fear conditioning leads to alterations in mRNAs levels 24 h after training in hippocampal neuropil, but not in hippocampal cell layers or BLA. The two main groups of altered mRNAs encode proteins involved in neuronal transmission, neuronal morphogenesis and neuronal development and the vast majority are known to be enriched in neurons. None of these mRNAs levels were altered by fear conditioning in EphB2lacZ/lacZ mice, which were also impaired in long-term fear memory. We show here that fear conditioning leads to an enduring alteration in mRNAs levels in hippocampal neuropil that is dependent on processes mediated by EphB2 that are needed for long-term memory formation.

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.

Investigating the effects of compound paralogous EPHB receptor mutations on mouse facial development.

BACKGROUND: Variation in facial shape may arise from the combinatorial or overlapping actions of paralogous genes. Given its many members, and overlapping expression and functions, the EPH receptor family is a compelling candidate source of craniofacial morphological variation. We performed a detailed morphometric analysis of an allelic series of E14.5 Ephb1-3 receptor mutants to determine the effect of each paralogous receptor gene on craniofacial morphology. RESULTS: We found that Ephb1, Ephb2, and Ephb3 genotypes significantly influenced facial shape, but Ephb1 effects were weaker than Ephb2 and Ephb3 effects. Ephb2-/- and Ephb3-/- mutations affected similar aspects of facial morphology, but Ephb3-/- mutants had additional facial shape effects. Craniofacial differences across the allelic series were largely consistent with predicted additive genetic effects. However, we identified a potentially important nonadditive effect where Ephb1 mutants displayed different morphologies depending on the combination of other Ephb paralogs present, where Ephb1+/- , Ephb1-/- , and Ephb1-/- ; Ephb3-/- mutants exhibited a consistent deviation from their predicted facial shapes. CONCLUSIONS: This study provides a detailed assessment of the effects of Ephb receptor gene paralogs on E14.5 mouse facial morphology and demonstrates how the loss of specific receptors contributes to facial dysmorphology.

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.

Inferred expression regulator activities suggest genes mediating cardiometabolic genetic signals.

Expression QTL (eQTL) analyses have suggested many genes mediating genome-wide association study (GWAS) signals but most GWAS signals still lack compelling explanatory genes. We have leveraged an adipose-specific gene regulatory network to infer expression regulator activities and phenotypic master regulators (MRs), which were used to detect activity QTLs (aQTLs) at cardiometabolic trait GWAS loci. Regulator activities were inferred with the VIPER algorithm that integrates enrichment of expected expression changes among a regulator's target genes with confidence in their regulator-target network interactions and target overlap between different regulators (i.e., pleiotropy). Phenotypic MRs were identified as those regulators whose activities were most important in predicting their respective phenotypes using random forest modeling. While eQTLs were typically more significant than aQTLs in cis, the opposite was true among candidate MRs in trans. Several GWAS loci colocalized with MR trans-eQTLs/aQTLs in the absence of colocalized cis-QTLs. Intriguingly, at the 1p36.1 BMI GWAS locus the EPHB2 cis-aQTL was stronger than its cis-eQTL and colocalized with the GWAS signal and 35 BMI MR trans-aQTLs, suggesting the GWAS signal may be mediated by effects on EPHB2 activity and its downstream effects on a network of BMI MRs. These MR and aQTL analyses represent systems genetic methods that may be broadly applied to supplement standard eQTL analyses for suggesting molecular effects mediating GWAS signals.

Plekhh1, a partner of myosin 1 and an effector of EphB2, controls the cortical actin network during cell repulsion.

EphB2-ephrinB signalling, which plays a major role in cell segregation during embryonic development and tissue homeostasis, induces an important reorganization of the cortical actin network. We have previously reported that myosin 1b contributes to reorganization of the cortical actin network upon EphB2 signalling. In this report, we identify Plekhh1 as a new partner of members of the myosin 1 family and EphB2 receptors. Plekhh1 interacts with myosin 1b via its N-terminal domain and with EphB2 via its C-terminal domain. Furthermore, Plekhh1 is tyrosine phosphorylated, and this depends on EphB2 kinase activity. Similar to the effects of manipulating levels of myosin 1b and myosin 1c, manipulation of Plekhh1 expression levels alters the formation of filopodia, the length of focal adhesions and the formation of blebs. Furthermore, binding of the Plekhh1 interacting domain to myosin 1b increases the motor activity of myosin 1b in vitro. Taken together, our data show that Plekhh1 is an effector of EphB2 and suggest that Plekhh1 regulates the cortical actin network via the interaction of its N-terminal domain with myosin 1 upon EphB2-ephrinB signalling.

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.

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.