EphB2 Signaling Research Articles

Androgen Receptor Promotes Lung Cancer Metastasis by Modifying the miR23a-3p/EPHB2 Pathway.

This study aimed to investigate the reasons behind the lower survival rates in male lung cancer patients than in female lung cancer patients. Through various techniques, such as Argonaute immunoprecipitation, luciferase assays, and ChIP, this study confirmed the positive effects of androgen receptor (AR) on lung cancer cell invasion across different in vitro cell lines and in vivo mouse models. The findings suggest that AR enhanced the invasion of lung cancer cells by modifying EPHB2 signals at the protein expression level, which in turn required changes in miRNA-23a-3p. Restoring miRNA-23a-3p could counteract the intensified invasion of lung cancer cells mediated by AR. This study revealed that AR may facilitate the lung cancer matastasis by modulating miRNA-23a-3p/EPHB2 signaling and that targeting this signaling pathway could provide new approaches to inhibit lung cancer metastasis.

EphB2 Signaling Is Implicated in Astrocyte-Mediated Parvalbumin Inhibitory Synapse Development.

Impaired inhibitory synapse development is suggested to drive neuronal hyperactivity in autism spectrum disorders (ASD) and epilepsy. We propose a novel mechanism by which astrocytes control the development of parvalbumin (PV)-specific inhibitory synapses in the hippocampus, implicating ephrin-B/EphB signaling. Here, we utilize genetic approaches to assess functional and structural connectivity between PV and pyramidal cells (PCs) through whole-cell patch-clamp electrophysiology, optogenetics, immunohistochemical analysis, and behaviors in male and female mice. While inhibitory synapse development is adversely affected by PV-specific expression of EphB2, a strong candidate ASD risk gene, astrocytic ephrin-B1 facilitates PV→PC connectivity through a mechanism involving EphB signaling in PV boutons. In contrast, the loss of astrocytic ephrin-B1 reduces PV→PC connectivity and inhibition, resulting in increased seizure susceptibility and an ASD-like phenotype. Our findings underscore the crucial role of astrocytes in regulating inhibitory circuit development and discover a new role of EphB2 receptors in PV-specific inhibitory synapse development.

EPEN-09. TARGETING EPHB2/ABL1 RESTORES ANTI-TUMOR IMMUNITY IN A PRECLINICAL MODEL OF EPENDYMOMA

Abstract BACKGROUND Ependymoma (EPN) is a childhood brain cancer that is often resistant to cytotoxic therapies. Molecular profiling has led to a better understanding of unique EPN subtypes and revealed a critical role of EPHB2 in driving disease. With this in mind, new treatments for EPN are desperately needed and should be developed in a molecular subtype-specific fashion. METHODS We developed a syngeneic mouse model of EPHB2-driven genetically engineered EPN tumor cells. We performed transcriptomic profiling and analysis of EPHB2-driven murine EPN tumors and human EPN datasets to reveal multiple protein kinases as potential druggable targets. After identifying the tyrosine kinase inhibitor, Dasatinib, as a potentially effective FDA-approved agent, we measured changes in the murine microenvironment during EPN growth and after Dasatinib treatment. RESULTS We determined that Dasatinib inhibited the growth of EPN both in vitro and in vivo, through blocking EPHB2 and ABL1 signaling. Furthermore, we identified an increased frequency of immunosuppressive M2-like tumor- associated macrophages (TAMs), which proportionally increased with tumor size during tumor progression. However, treatment with Dasatinib reprogrammed the EPN immune microenvironment by polarizing TAMs toward an anti-tumor M1-like phenotype and increasing CD8 T cell activation. In addition, Dasatinib treatment induced complete regression of established EPN tumors in 78% of the animals and protected survivors against tumor recurrence. Depletion of CD8 T cells compromised the durability of response and reduced overall survival. CONCLUSION In conclusion, these data indicate that Dasatinib may be an effective therapy for EPHB2-driven molecular subgroup of EPN by activating the anti-tumor immune response and support further investigation of Dasatinib in clinical trials.

Abstract PR-004: Targeting EPHB2/ABL1 restores anti-tumor immunity in a preclinical model of ependymoma

Abstract Purpose: Ependymoma (EPN) is a common type of brain tumor in children and is often resistant to available cytotoxic therapies. Molecular profiling studies have led to a better understanding of unique EPN subtypes and revealed a critical role of EPHB2. However, the immune system's role in tumor progression and treatment response remains poorly understood. New treatments for EPN are desperately needed and should be developed in a molecular subtype-specific fashion. Experimental Design: We developed a syngeneic mouse model using subtype-specific EPHB2-driven genetically engineered EPN tumor cells. Druggable targets were identified by matching transcriptomic signatures with the target spectrum of FDA-approved drugs. After identifying dasatinib as a potentially effective agent, we measured the changes in the immune microenvironment during EPN growth and after dasatinib treatment. Results: Transcriptomic profiling of EPHB2-driven EPN and analysis of a human EPN dataset revealed multiple protein kinases as potential druggable targets. We found that the multikinase inhibitor dasatinib potently inhibited the growth of EPN both in vitro and in vivo, mainly through blocking EPHB2 and ABL1 signaling. We found an increased frequency of immunosuppressive M2-like tumor-associated macrophages (TAMs), which proportionally increased with tumor size during tumor progression. However, treatment with dasatinib reprogrammed the EPN immune microenvironment by polarizing the TAMs toward an anti-tumor M1-like phenotype and increasing CD8 T cell activation. Furthermore, dasatinib treatment induced complete regression of established EPN tumors in 78% of the animals and protected survivors against tumor recurrence. Depletion of CD8 T cells compromised the durability of EPN responses and reduced overall survival. Conclusions: These data indicate that dasatinib may be an effective therapy for EPHB2-driven molecular subgroup of EPNs by activating the anti-tumor immune response and support further investigations of dasatinib in clinical trials. Citation Format: Taylor P. Uccello, Jun Ren, Zohreh Amoozgar, Yuhui Zhao, Pin-Ji Lei, William W. Ho, Sylvie Roberge, Peigen Huang, Dan G. Duda, Lei Xu, Rakesh K. Jain. Targeting EPHB2/ABL1 restores anti-tumor immunity in a preclinical model of ependymoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-004.

The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett’s Neoplasia

Background & AimsMechanisms contributing to the onset and progression of Barrett’s (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett’s neoplasia. MethodsWhole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. ResultsThe vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. ConclusionsThe EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

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.

EphrinB2-EphB2 signaling for dendrite protection after neuronal ischemia in vivo and oxygen-glucose deprivation in vitro

In order to rescue neuronal function, neuroprotection should be required not only for the neuron soma but also the dendrites. Here, we propose the hypothesis that ephrin-B2-EphB2 signaling may be involved in dendritic degeneration after ischemic injury. A mouse model of focal cerebral ischemia with middle cerebral artery occlusion (MCAO) method was used for EphB2 signaling test in vivo. Primary cortical neuron culture and oxygen-glucose deprivation were used to assess EphB2 signaling in vitro. siRNA and soluble ephrin-B2 ectodomain were used to block ephrin-B2-Ephb2 signaling. In the mouse model of focal cerebral ischemia and in neurons subjected to oxygen-glucose deprivation, clustering of ephrin-B2 with its receptor EphB2 was detected. Phosphorylation of EphB2 suggested activation of this signaling pathway. RNA silencing of EphB2 prevented neuronal death and preserved dendritic length. To assess therapeutic potential, we compared the soluble EphB2 ectodomain with the NMDA antagonist MK801 in neurons after oxygen-glucose deprivation. Both agents equally reduced lactate dehydrogenase release as a general marker of neurotoxicity. However, only soluble EphB2 ectodomain protected the dendrites. These findings provide a proof of concept that ephrin-B2-EphB2 signaling may represent a novel therapeutic target to protect both the neuron soma as well as dendrites against ischemic injury.

Therapeutic effects of ephrin B receptor 2 inhibitors screened by molecular docking on cutaneous squamous cell carcinoma

Background Cutaneous squamous cell carcinoma (CSCC) is the most known form type of metastatic skin cancer. Activation of ephrin B receptor 2 (EphB2) signaling can promote the metastasis, invasion, and angiogenesis of CSCC cells. Therefore, EphB2 may act as a therapeutic target for CSCC. Here, we screened the inhibitors for EphB2 using molecular docking and then evaluated the effects of the identified inhibitors on cancer-related features of CSCC cells. Methods The Schrodinger docking tool was used to predict the three-dimensional structure of EphB2 protein and its ligand binding sites, and EphB2 inhibitors were screened by high-throughput virtual screening combined with molecular docking. The effects of EphB2 inhibitors were analyzed for cell viability, proliferation, apoptosis, migration, invasion, and xenograft tumor growth. Results In vitro experiments, the identified small-molecule inhibitors markedly inhibited the skin cancer cells proliferation, induced apoptosis, altered the cell cycle, and inhibited cell invasion and migration in our study. In a xenograft model, the identified small-molecule inhibitors induced changes in the epithelial mesenchymal transition, which affected the progression of CSCC. Conclusion EphB2 small-molecule inhibitors had anti-CSCC effects, establishing a solid theoretical basis for clinical research.

TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response

The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.

MP51-07 LOSS OF EPHRIN SIGNALING ALTERS THE LIPID RHEOSTAT ASSOCIATED WITH PROSTATE CANCER RACIAL DISPARITIES

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-07 LOSS OF EPHRIN SIGNALING ALTERS THE LIPID RHEOSTAT ASSOCIATED WITH PROSTATE CANCER RACIAL DISPARITIES Alejandro Morales, Victoria Gil, Susan Crawford, Simon Hayward, and Omar Franco* Alejandro MoralesAlejandro Morales More articles by this author , Victoria GilVictoria Gil More articles by this author , Susan CrawfordSusan Crawford More articles by this author , Simon HaywardSimon Hayward More articles by this author , and Omar Franco*Omar Franco* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The presence of abdominal central obesity in prostate cancer (PCa) patients is associated with aggressive tumors. Compared to Caucasians (CA), African American (AA) men are at highest risk of developing and dying from PCa. The Center for Disease Control reports racial disparities in adult obesity (38.4% AA vs. 28.6% in CA). In an obese environment, cancer cells store fatty acids in lipid droplets (LD), composed of triacylglycerols (TAG) and cholesterol esters, which can be used as an energy source to sustain proliferation and migration. Decreased gene expression of members of the ephrin family have been linked to PCa-RD. Here we show the functional consequences of aberrant ephrin signaling in lipid metabolism on PCa tumorigenesis in AA compared to CA. METHODS: The density and size of LD were quantified in PCa specimens from different racial backgrounds. EPHB2 signaling was manipulated in PCa cells using siRNA and CRISPR approaches. LD were quantified under basal and obesogenic (added oleic acid) environments. Molecules involved in TAG metabolism were assessed. The effects of EPHB2 loss on PCa cells growth and local invasion was tested in vitro and in vivo. RESULTS: LD density increased in high grade tumors. Under obesogenic conditions, PCa cells from AA showed increased proliferation compared to CA. Loss of EPHB2 increased the tumorigenicity of PCa cells. Quantification of LD profiles showed racial differences. Alteration of EPHB2 led to differential expression of TAG-associated molecules DGAT1, DGAT2 and ATGL in AA compared to CA. Targeting key TAG molecules associated with EPHB2-induced effects reduced the tumorigenicity of PCa cells. Centrosome amplification and microtubule organizing center (MTOC) were associated with lipid metabolic changes in cells with decreased EphB2 signaling. CONCLUSIONS: Loss of EPHB2 signaling in PCa cells triggers aberrant lipid metabolism leading to excessive accumulation of LD. Because loss of ephrin signaling is associated with PCa-RD, this lipid-rich energy status may serve as a source of energy to fuel a more aggressive phenotype observed in AA. Several TAG inhibitors are currently being evaluated in clinical trials as anti-obesity agents. The potential of these drugs to elicit anti-tumorigenic utility in patients with altered ephrin levels requires further study. Source of Funding: R01 CA242920-01 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e766-e766 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alejandro Morales More articles by this author Victoria Gil More articles by this author Susan Crawford More articles by this author Simon Hayward More articles by this author Omar Franco* More articles by this author Expand All Advertisement PDF downloadLoading ...

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.

Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer.

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

EphB2 signaling-mediated Sirt3 expression reduces MSC senescence by maintaining mitochondrial ROS homeostasis

Disruption of mitochondrial reactive oxygen species (mtROS) homeostasis is a key factor inducing UCB-MSC senescence. Accordingly, preventing mtROS accumulation will help in suppressing the UCB-MSC senescence. In this study, we observed that the expressions of EphrinB2 and EphB2 were inversely regulated by UCB-MSC passage-dependent manner. EphB2 signaling induced mitochondrial translocation of Sirt3. The knockdown of SIRT3 inhibited the effect of EphB2 signaling in UCB-MSCs. Subsequently, EphrinB2-Fc induced the nuclear translocation of Nrf-2 via c-Src phosphorylation dependent manner, and Sirt3 expression was regulated by Nrf-2. Among Sirt3 target genes, EphB2 signaling increased MnSOD and reduced the mtROS level in UCB-MSCs. Furthermore, the deacetylase effect of Sirt3 enhanced the MnSOD activity by deacetylation at the lysine 68 residue and therapeutic effect of UCB-MSCs on skin-wound healing was increased by EphB2 activation. In conclusion, the EphB2 can serve as a novel target for the optimizing the therapeutic use of UCB-MSCs in wound repair by MnSOD-mediated mtROS scavenging through EphB2/c-Src signaling pathway and Nrf-2-dependent Sirt3 expression.

TNF‐α/TNFR2 Regulatory Axis Stimulates EphB2‐Mediated Neuroregeneration Via Activation of NF‐κB

HIV-1 infected individuals are at high risk of developing HIV-associated neurocognitive disorders (HAND) as HIV infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxic effects of HIV-1 are primarily a result of viral replication leading to the production of inflammatory chemokines and cytokines, including TNF-α. Given an important role of TNF-α in regulating synaptic plasticity, we investigated the effects of TNF-α on the development of neuronal processes after mechanical injury, and we showed that TNF-α treatment stimulates the regrowth of neuronal processes. To investigate transcriptional effects of TNF-α on synaptic plasticity, we analyzed both human neurosphere and isolated neuronal cultures for the regulation of genes central to synaptic alterations during learning and memory. TNF-α treatment upregulated Ephrin receptor B2 (EphB2), which is strongly involved in dendritic arborization and synaptic integrity. TNF-α strongly activates the NF-κB pathway, therefore, we propose that TNF-α-induced neurite regrowth occurs primarily through EphB2 signaling via stimulation of NF-κB. EphB2 promoter activity increased with TNF-α treatment and overexpression of NF-κB. Direct binding of NF-κB to the EphB2 promoter occurred in the ChIP assay, and site-directed mutagenesis identified binding sites involved in TNF-α-induced EphB2 activation. TNF-α induction of EphB2 was determined to occur specifically through TNF-α receptor 2 (TNFR2) activation in human primary fetal neurons. Our observations provide a new avenue for the investigation on the impact of TNF-α in the context of HIV-1 neuronal cell damage as well as providing a potential therapeutic target in TNFR2 activation of EphB2.

Infralimbic EphB2 Modulates Fear Extinction in Adolescent Rats.

Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase.

Myosin 1b functions as an effector of EphB signaling to control cell repulsion.

Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.

EphB2 Promotes Progression of Cutaneous Squamous Cell Carcinoma

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell-specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer.

Optogenetic Control of EphB Signaling in Dendrites

EphrinB receptors (EphBs) are receptor tyrosine kinases that promote dendritic spine morphogenesis by altering local actin polymerization at axo‐dendritic contacts. Soluble ligands have been used with neuronal cultures to activate EphBs, though local stimulation is more desirable to model local contact and study downstream signaling dynamics. Since EphB signaling requires receptor clustering, we designed an optogenetic tool, named optoEphB, based on an optimized mutant of the plant protein cryptochrome 2 (Cry2olig, residues 1‐498 with E490G mutation), which shows blue light‐induced clustering. OptoEphB consists of the EphB2 cytoplasmic domain fused to Cry2olig and mCherry at the C‐terminus with myristoylation at the N‐terminus. Global and focal blue light illumination of HEK293 cells expressing optoEphB showed reversible optoEphB clustering and retraction of membrane protrusions, indicative of EphB2 signaling. In hippocampal neurons, focal illumination of dendrites produced transient, dynamic protrusions that resembled dendritic filopodia. We observed recruitment of the pleckstrin homology domain of Akt in illuminated regions, suggesting phosphoinositide 3‐kinase (PI3K) activation in this process. Our results demonstrate that optoEphB locally activates EphB signaling and thus represents a novel optogenetic method of Eph receptor activation. Further experiments will determine PI3K‐dependence of filopodial protrusions and use biosensors to measure the spatiotemporal dynamics of downstream Rho GTPase activity, implicated in EphB‐driven spine morphogenesis. These results will bring new insights into current models of dendritic spine development.

EphB2 regulates contact-dependent and contact-independent signaling to control platelet function

AbstractThe Eph kinases, EphA4 and EphB1, and their ligand, ephrinB1, have been previously reported to be present in platelets where they contribute to thrombus stability. Although thrombus formation allows for Eph-ephrin engagement and bidirectional signaling, the importance specifically of Eph kinase or ephrin signaling in regulating platelet function remained unidentified. In the present study, a genetic approach was used in mice to establish the contribution of signaling orchestrated by the cytoplasmic domain of EphB2 (a newly discovered Eph kinase in platelets) in platelet activation and thrombus formation. We conclude that EphB2 signaling is involved in the regulation of thrombus formation and clot retraction. Furthermore, the cytoplasmic tail of this Eph kinase regulates initial platelet activation in a contact-independent manner in the absence of Eph-ephrin ligation between platelets. Together, these data demonstrate that EphB2 signaling not only modulates platelet function within a thrombus but is also involved in the regulation of the function of isolated platelets in a contact-independent manner.