Ephrin Ligands Research Articles

Limited importance of EphrinA1–ligand, Src kinase, and focal adhesion kinase in EphA2-mediated regulation of metastasis in Mel-Juso and A375 human melanoma cells

Limited importance of EphrinA1–ligand, Src kinase, and focal adhesion kinase in EphA2-mediated regulation of metastasis in Mel-Juso and A375 human melanoma cells

361 Ephrin-A1 ligand strengthens tight junction function and prevents Th2 cytokine-induced barrier collapse

Atopic dermatitis (AD) is a chronic skin disease affecting all ages at a worldwide prevalence rate up to 20%. AD is largely driven by immune signals initiated by Th2 cytokines including interleukin (IL)-4 and IL-13. However, mounting evidence points to a central role for epidermal barrier dysfunction in the pathogenesis of AD. We have shown that the epidermal tight junction (TJ) network, a critical component of the barrier, depends on the receptor tyrosine kinase EphA2. Therefore, we hypothesize that EphA2 is a positive regulator of TJs in epidermis and can be targeted to improve epidermal barrier function. In 2D keratinocyte cultures, engaging EphA2 with its ligand ephrin-A1 enhances TJ barrier function compared to control (P=0.0001), as measured by trans-epithelial electrical resistance (TEER). This enhancement is accompanied by the immunofluorescent accumulation of TJ proteins, but only small changes in protein and transcript expression. Increased TEER (P<0.05) and TJ formation are also seen in 3D human epidermal equivalents (3D HEE) after ephrin-A1 treatment. These results led us to ask if ephrin-A1 can prevent epidermal barrier defects in in vitro models of AD. Concurrent treatment with IL-4 and IL-13 induces an AD-like phenotype apparent by changes in epidermal morphology. We show that ephrin-A1 treatment in 3D human skin equivalents (3D HSE) restores keratohyalin granules and the stratum corneum which are lost in 3D HSE after cytokine treatment alone. In human explant cultures of truncal skin, IL-4+IL-13 cause epidermal spongiosis and increased epidermal thickness, effects that are abrogated by ephrin-A1. In addition to altering epidermal morphology, we find that IL-4+IL-13 reduce TEER in 3D HEE compared to controls (P<0.05) and obliterate TJs, shown by loss of occludin immunofluorescence. This cytokine-induced catastrophe of TJ function and formation was rescued by co-treatment with ephrin-A1. Taken together, these results suggest that targeting the EphA2 signaling axis not only enhances TJs, but can also restore epidermal barrier function in the face of Th2 cytokine-driven TJ disassembly.

Ephrins and Eph Receptor Signaling in Tissue Repair and Fibrosis.

Fibrosis is a pathological feature of many human diseases that affect multiple organs. The development of anti-fibrotic therapies has been a difficult endeavor due to the complexity of signaling pathways associated with fibrogenic processes, complicating the identification and modulation of specific targets. Evidence suggests that ephrin ligands and Eph receptors are crucial signaling molecules that contribute to physiological wound repair and the development of tissue fibrosis. Here, we discuss recent advances in the understanding of ephrin and Eph signaling in tissue repair and fibrosis. Ephrin-B2 is implicated in fibrosis of multiple organs. Intercepting its signaling may help counteract fibrosis. Ephrins and Eph receptors are candidate mediators of fibrosis. Ephrin-B2, in particular, promotes fibrogenic processes in multiple organs. Thus, therapeutic strategies targeting Ephrin-B2 signaling could yield new ways to treat organ fibrosis.

Ephrin-B2 inhibits Aβ25-35-induced apoptosis by alleviating endoplasmic reticulum stress and promoting autophagy in HT22 cells

Previous studies have shown that Erythropoietin-producing hepatocellular carcinoma receptor B2 (EphB2) inhibited Aβ-induced neuron apoptosis and improved cognition in AD mice, but the role of which ligand Ephrin B2 (Ephrin-B2, efnB2) is not clear. The aim of this study was to investigate the effect of the efnB2-activated Eph/efn forward signaling pathway on Aβ-induced HT22 hippocampal cell apoptosis using recombination mouse Ephrin B2-Fc chimera protein (efnB2-Fc).We found that non-toxic concentrations of efnB2-Fc decreased the release of lactate dehydrogenase (LDH) in HT22 cells and reduced cell apoptosis in a dose-dependent manner. Further studies revealed that efnB2-Fc alleviated Aβ-induced ER stress and decreased the expression of ER-stress-related transcriptional factor C/EBP homologous protein (CHOP), binding immunoglobulin protein (Bip) and phosphorylated eukaryotic translation initiation factor 2 subunit α (p-eIF2α) in HT22 cells. These effects of efnB2-Fc were related to the inhibition of Akt/mTOR signal transduction, an increase in the level of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ), and the activation of the autophagy pathway. Our results indicate that the efnB2-mediated forward signaling pathway may activate the autophagy pathway to alleviate the Aβ-induced ER stress and apoptosis in the HT22 cell.

Dynamic Alterations of Retinal EphA5 Expression in Retinocollicular Map Plasticity.

The topographically ordered retinocollicular projection is an excellent system for studying the mechanism of axon guidance. Gradients of EphA receptors in the retina and ephrin-As in the superior colliculus (SC) pattern the anteroposterior axis of the retinocollicular map, but whether they are involved in map plasticity after injury is unknown. Partial damage to the caudal SC at birth creates a compressed, complete retinotopic map in the remaining SC without affecting visual response properties. Previously, we found that the gradient of ephrin-A expression in compressed maps is steeper than normal, suggesting an instructive role in compression. Here we measured EphA5 mRNA and protein levels after caudal SC damage in order to test the hypothesis that changes in retinal EphA5 expression occur that are complementary to the changes in collicular ephrin-A expression. We find that the nasotemporal gradient of EphA5 receptor expression steepens in the retina and overall expression levels change dynamically, especially in temporal retina, supporting the hypothesis. This change in receptor expression occurs after the change in ephrin-A ligand expression. We propose that changes in the retinal EphA5 gradient guide recovery of the retinocollicular projection from early injury. This could occur directly through the change in EphA5 expression instructing retino-SC map compression, or through ephrin-A ligand signaling instructing a change in EphA5 receptor expression that in turn signals the retinocollicular map to compress. Understanding what molecular signals direct compensation for injury is essential to developing rehabilitative strategies and maximizing the potential for recovery.

Silencing of EPHB2 promotes the epithelial-mesenchymal transition of skin squamous cell carcinoma-derived A431 cells.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligand ephrins serve crucial roles in the interactions among epithelial cells. Eph receptor/ephrin signaling regulates cell functions, including proliferation, differentiation and migration, via these cell-cell interactions. We reported previously that EPHB2, a member of the Eph receptor family, was highly expressed in chemically induced cutaneous squamous cell carcinoma (cSCC) tissues in mice. Although the higher expression level of EPHB2 has been observed in various human cancers, its roles in the development and progression of cancers are still unclear. In the present study, the functional implications of EPHB2 in the acquisition of malignant phenotypes of cSCC cells was investigated. Silencing of EPHB2 in the human cSCC cell line A431 induced epithelial-mesenchymal transition (EMT)-like morphological changes accompanied by a significant upregulation of epithelial-mesenchymal transition-associated genes such as zinc finger E-box binding homeobox 1/2. In addition, silencing of EPHB2 suppressed anchorage-independent cell growth under 3D culture conditions. Consistent with these observations, EPHB2 exhibited higher levels of expression in tumor spheres formed under 3D culture conditions than in cells cultured in adherent form, and the expression pattern of EMT markers indicated that EMT was suppressed in tumor spheres. The results of the present study indicated that EPHB2 serves a pivotal role in promoting the anchorage-independent growth of A431 cells through the suppression of EMT.

Rs12904 polymorphism in the 3'-untranslated region of ephrin A1 ligand and the risk of sporadic colorectal cancer in the Iranian population.

Colorectal cancer (CRC) is rated as the second cause of cancer death. Genetic determinants are considered as driving forces in the development of sporadic CRC. Single-nucleotide polymorphisms (SNPs), due to their abundance in the human genome with collectively huge effect on cellular signaling pathways, are attributed as the main genetic factor in disease susceptibility including cancers. MicroRNAs are contributing to posttranslational gene regulation. They exert their regulatory function by binding to their specific recognition sequences located at 3'-untranslated region (UTR) of mRNAs. In the present study, we have elucidated the role of rs12904, a naturally occurring SNP, in the recognition site of miR200c in the 3'UTR of ephrin A1 ligand gene, in the development of sporadic CRC in the Iranian population. A case-control study using 152 CRC patients and 160 noncancerous counterparts was conducted to determine the rs12904 genotypes using polymerase chain reaction-restriction fragment length polymorphism method. The results revealed no significant association between the rs12904 and sporadic CRC (odds ratio = 0.97, 95% confidence interval = 0.70-1.34). The frequency of genotypes and also alleles of the mentioned polymorphism were not significantly different between case and control groups (P = 0.765 and P = 0.847, respectively). The results suggest that this polymorphism probably has not a crucial role in the Iranian CRC risk and is not an important potential risk factor in molecular diagnostics of mentioned disease among the Iranian population.

Targeted delivery of YSA-functionalized and non-functionalized polymeric nanoparticles to injured pulmonary vasculature

Ephrin type-A receptor 2 (EphA2) is a transmembrane receptor which is upregulated in injured lungs, including those treated with bleomycin. YSA peptide (YSAYPDSVPMMS), a mimic of ephrin ligands, binds to EphA2 receptors on cell surface with high affinity. In this study, we assessed the ability of YSA-functionalized and non-functionalized poly (dl-lactide-co-glycolide) (PLGA) nanoparticles to enhance delivery to bleomycin treated cultured vascular endothelial cells and, in a bleomycin induced lung injury mouse model. Nanoparticles were loaded with a lipophilic fluorescent dye. Human umbilical vein endothelial cells (HUVEC) with or without 2-day bleomycin pretreatment (25 µg/ml) and adult mice with or without intratracheal instillation of bleomycin (0.1 U) were dosed with nanoparticles. Mice received nanoparticles via tail vein injection 4 days after bleomycin treatment. Three days after nanoparticle injection, tissues (lung, heart, kidney, spleen, liver, brain, eyes and whole blood) were harvested and quantified for fluorescence using IVIS imaging. Mean particle uptake increased with time and concentration for both types of particles in HUVEC, with the uptake being higher for YSA-functionalized nanoparticles. Bleomycin treatment increased the 3-h uptake of both types of nanoparticles in HUVEC by about two-fold, with the YSA-functionalized nanoparticle uptake being 1.66-fold compared to non-functionalized nanoparticles (p < .05). In mice, bleomycin injury resulted in 2.3- and 4.7-fold increase in the lung levels of non-functionalized and YSA-functionalized nanoparticles (p < .05), respectively, although the differences between the two particle types were not significant. In conclusion, PLGA nanoparticle delivery to cultured vascular endothelial cells and mouse lungs in vivo is higher following bleomycin treatment, with the delivery tending to be higher for YSA functionalized nanoparticles.

Changes in ephrin gene expression during bone healing identify a restricted repertoire of ephrinsmediating fracture repair.

To identify the repertoire of ephrin genes that might regulate endochondral bone fracture repair, we examined changes in ephrin ligand and receptor (Eph) gene expression in fracture callus tissues during bone fracture healing. Ephrin and Eph proteins were then localized in the fracture callus tissues present when changes in gene expression were observed. Ephrin gene expression was widespread in fracture tissues, but the repertoire of ephrin genes with significant changes in expression that might suggest a regulatory role in fracture callus development was restricted to the ephrin A family members Epha4, Epha5 and the ephrin B family member Efnb1. After 3 weeks of healing, Epha4 fracture expression was downregulated from 1.3- to 0.8-fold and Epha5 fracture expression was upregulated from 1.2- to 1.5-fold of intact contralateral femur expression, respectively. Efnb1 expression was downregulated from 1.5- to 1.2-fold after 2 weeks post-fracture. These ephrin proteins were localized to fracture callus prehypertrophic chondrocytes and osteoblasts, as well as to the periosteum and fibrous tissues. The observed positive correlation between mRNA levels of EfnB1 with Col10 and Epha5 with Bglap, together with colocalized expression with their respective proteins, suggest that EfnB1 is a positive mediator of prehypertrophic chondrocyte development and that Epha5 contributes to osteoblast-mediated mineralization of fracture callus. In contrast, mRNA levels of Epha4 and Efnb1 correlated negatively with Bglap, thus suggesting a negative role for these two ephrin family members in mature osteoblast functions. Given the number of family members and widespread expression of the ephrins, a characterization of changes in ephrin gene expression provides a basis for identifying ephrin family members that might regulate the molecular pathways of bone fracture repair. This approach suggests that a highly restricted repertoire of ephrins, EfnB1 and EphA5, are the major mediators of fracture callus cartilage hypertrophy and ossification, respectively, and proposes candidates for additional functional study and eventual therapeutic application.

Regulation of islet glucagon secretion: Beyond calcium.

The islet of Langerhans plays a key role in glucose homeostasis through regulated secretion of the hormones insulin and glucagon. Islet research has focused on the insulin-secreting β-cells, even though aberrant glucagon secretion from α-cells also contributes to the aetiology of diabetes. Despite its importance, the mechanisms controlling glucagon secretion remain controversial. Proper α-cell function requires the islet milieu, where β- and δ-cells drive and constrain α-cell dynamics. The response of glucagon to glucose is similar between isolated islets and that measured in vivo, so it appears that the glucose dependence requires only islet-intrinsic factors and not input from blood flow or the nervous system. Elevated intracellular free Ca2+ is needed for α-cell exocytosis, but interpreting Ca2+ data is tricky since it is heterogeneous among α-cells at all physiological glucose levels. Total Ca2+ activity in α-cells increases slightly with glucose, so Ca2+ may serve a permissive, rather than regulatory, role in glucagon secretion. On the other hand, cAMP is a more promising candidate for controlling glucagon secretion and is itself driven by paracrine signalling from β- and δ-cells. Another pathway, juxtacrine signalling through the α-cell EphA receptors, stimulated by β-cell ephrin ligands, leads to a tonic inhibition of glucagon secretion. We discuss potential combinations of Ca2+ , cAMP, paracrine and juxtacrine factors in the regulation of glucagon secretion, focusing on recent data in the literature that might unify the field towards a quantitative understanding of α-cell function.

Abnormal expression of ephrin-A5 affects brain development of congenital hypothyroidism rats.

EphA5 and its ligand ephrin-A5 interaction can trigger synaptogenesis during early hippocampus development. We have previously reported that abnormal EphA5 expression can result in synaptogenesis disorder in congenital hypothyroidism (CH) rats. To better understand its precise molecular mechanism, we further analyzed the characteristics of ephrin-A5 expression in the hippocampus of CH rats. Our study revealed that ephrin-A5 expression was downregulated by thyroid hormone deficiency in the developing hippocampus and hippocampal neurons in rats. Thyroxine treatment for hypothyroid hippocampus and triiodothyronine treatment for hypothyroid hippocampal neurons significantly improved ephrin-A5 expression but could not restore its expression to control levels. Hypothyroid hippocampal neurons in-vitro showed synaptogenesis disorder characterized by a reduction in the number and length of neurites. Furthermore, the synaptogenesis-associated molecular expressions of NMDAR-1 (NR1), PSD95 and CaMKII were all downregulated correspondingly. These results suggest that ephrin-A5 expression may be decreased in CH, and abnormal activation of ephrin-A5/EphA5 signaling affects synaptogenesis during brain development. Such findings provide an important basis for exploring the pathogenesis of CH genetically.

Compartments with predominant ephrin-B1 and EphB2/B4 expression are present alternately along the excurrent duct system in the adult mouse testis and epididymis.

Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice. mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively. Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis. Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis.

EphA3 contributes to tumor growth and angiogenesis in human gastric cancer cells.

Eph receptor tyrosine kinases and their ephrin ligands, mediate an important cell communication system both in normal and oncogenic development, and play central roles in a series of processes including angiogenesis, stem cell maintenance and cancer metastasis. Eph receptor A3 (EphA3), commonly overexpressed in a broad range of cancers, including gastric cancer (GC), is related to tumor progression. Our previous study revealed that EphA3 may play important roles in tumorigenesis and angiogenesis in GC. However, its exact role and the mechanisms underlying its function in GC remain unclear. In the present study, lentivirus‑mediated RNA interference was employed to knock down the expression of EphA3 in GC HGC‑27 cells. Functional analyses indicated that depletion of EphA3 expression inhibited the cell growth and tumorigenicity of HGC‑27 cells invitro and invivo. Furthermore, knockdown of the expression of EphA3 in HGC‑27 cells inhibited tube formation and migration of HUVEC endothelial cells. Tumor angiogenesis invivo was also inhibited upon EphA3 knockdown in HGC‑27 cells, with reduced microvessel density (MVD) in xenograft models. We further revealed that EphA3 depletion inhibited tumor angiogenesis and migration through the signal transducer and activator of transcription 3/vascular endothelial growth factor (STAT3/VEGF) signaling pathway. These results indicated that EphA3 may be an effective prognostic indicator and a potential target for GC therapy.

Prognostic Significance of EPHB2 Expression in Colorectal Cancer Progression.

BackgroundA receptor tyrosine kinase for ephrin ligands, EPHB2, is expressed in normal colorectal tissues and colorectal cancers (CRCs). The aim of this study was to investigate EPHB2 expression over CRC progression and determine its prognostic significance in CRC.MethodsTo measure EPHB2 mRNA and protein expression, real-time polymerase chain reaction and immunohistochemistry were performed in 32 fresh-frozen and 567 formalin-fixed paraffin-embedded CRC samples, respectively. We further investigated clinicopathological features and overall and recurrence-free survival according to EPHB2 protein expression.ResultsThe EPHB2 level was upregulated in CRC samples compared to non-cancerous tissue in most samples and showed a strong positive correlation with AXIN2. Notably, CD44 had a positive association with both mRNA and protein levels of EPHB2. Immunohistochemical analysis revealed no difference in EPHB2 expression between adenoma and carcinoma areas. Although EPHB2 expression was slightly lower in invasive fronts compared to surface area (p < .05), there was no difference between superficial and metastatic areas. EPHB2 positivity was associated with lymphatic (p < .001) and venous (p = .001) invasion, TNM stage (p < .001), and microsatellite instability (p = .036). Kaplan–Meier analysis demonstrated that CRC patients with EPHB2 positivity showed better clinical outcomes in both overall (p = .049) and recurrence-free survival (p = .015). However, multivariate analysis failed to show that EPHB2 is an independent prognostic marker in CRCs (hazard ratio, 0.692; p = .692).ConclusionsOur results suggest that EPHB2 is overexpressed in a subset of CRCs and is a significant prognostic marker.

Eph Receptor Effector Ephexin Mediates Olfactory Dendrite Targeting in Drosophila.

Deciphering the mechanisms of sensory neural map formation is a central aim in neurosciences. Failure to form a correct map frequently leads to defects in sensory processing and perception. The olfactory map develops in subsequent steps initially forming a rough and later a precise map of glomeruli in the antennal lobe (AL), mainly consisting of olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites. The mechanisms underpinning the later stage of class-specific glomerulus formation are not understood. Recent studies have shown that the important guidance molecule Eph and its ligand ephrin play a role in class-specific PN targeting. Here, we reveal aspects of the mechanism downstream of Eph signaling during olfactory map formation. We show that the Eph-specific RhoGEF Ephexin (Exn) is required to fine tune PN dendrite patterning within specific glomeruli. We provide the first report showing an in vivo neurite guidance defect in an exn mutant. Interestingly, the quality of the phenotypes is different between eph and exn mutants; while loss of Eph leads to strong misprojections of DM3/Or47a neurons along the medial-lateral axis of the antennal lobe (AL), loss of Exn induces ventral ectopic innervation of a neighboring glomerulus. Genetic interaction experiments suggest that differential signaling of the small GTPases Rac1 and Cdc42 mediated by Exn-dependent and -independent Eph signaling fine tunes spatial targeting of PN dendrites within the olfactory map. We propose that their distinct activities on the actin cytoskeleton are required for precise navigation of PN dendrites within the olfactory map. Taken together, our results suggest that the precise connectivity of an individual neuron can depend on different modes of signaling downstream of a single guidance receptor. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.

Activation of EphA4 induced by EphrinA1 exacerbates disruption of the blood-brain barrier following cerebral ischemia-reperfusion via the Rho/ROCK signaling pathway.

Vascular dementia (VD) is a syndrome characterized by progressive cognitive decline. According to previous studies, stroke is considered to be a risk factor for VD. The disruption of the blood-brain barrier (BBB) is pivotal to the pathology of stroke, as it contributes to post-stroke inflammation and edema. It has been reported that the Eph/Ephrin signaling pathway serves an important role in central nervous system injury. However, the role of EphrinA1/EphA4 signaling in BBB damage following ischemic stroke has not yet been reported. Oxygen-glucose deprivation/reperfusion was performed to detect changes in EphrinA1 and EphA4 expression in human brain microvascular endothelial cells (HBMECs). Male mice were randomly divided into four groups [Sham, ischemia-reperfusion (I/R), I/R+EphrinA1 and I/R+EphA4] to observe the role of EphrinA1 and EphA4 under I/R conditions in vivo. The results of the present study revealed that the expression of EphrinA1 and EphA4 was significantly increased following I/R in vitro and in vivo. The administration of soluble ligand EphrinA1 enhanced CD68+ cell accumulation, brain edema and dysfunction of the BBB, with lower expression levels of zonula occludens-1 (ZO-1) and Claudin-5. In addition, EphrinA1-treated mice had a higher level of caspase-3 and a lower level of phosphorylated-protein kinase B. However, the effects of EphrinA1 were abolished by EphA4-Fc, an inhibitor of EphA4. These results suggested that EphrinA1 exerted its effects on I/R injury via the activated EphA4 receptor. In addition, EphrinA1 decreased ZO-1 and Claudin-5 expression through the Rho/Rho associated kinase (ROCK) signaling pathway, which was attenuated by the pharmacological inhibition of Rho (C3 transferase) or ROCK (Y-27632). In conclusion, the present study provides evidence that the activation of EphA4 induced by EphrinA1 contributes to BBB damage following ischemic stroke through the Rho/ROCK signaling pathway, which highlights a potential therapeutic strategy for ischemic stroke and may help the development of preventative interventions for VD.

Epigenetic Control of the Notch and Eph Signaling Pathways by the Prion Protein: Implications for Prion Diseases.

Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrPC. We further show that both prion infection and PrPC depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrPC-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.

Cancer-associated fibroblasts promote gastric tumorigenesis through EphA2 activation in a ligand-independent manner.

Under physiologic conditions, the binding of erythropoietin-producing hepatocellular (Eph) A2 receptor and its ligand ephrinA1 results in decreased EphA2 level and tumor suppression. However, EphA2 and ephrinA1 are highly expressed in human cancers including gastric adenocarcinoma. In this study, we tested our hypothesis that cancer-associated fibroblasts (CAFs) promote gastric tumorigenesis through EphA2 signaling in a ligand-independent manner. Expression of EphA2 protein in primary tumor tissues of 91 patients who underwent curative surgery for gastric adenocarcinoma was evaluated by immunohistochemistry and western blotting. Conditioned medium of cancer-associated fibroblasts (CAF-CM) was used to evaluate the tumorigenic effect of CAFs on gastric cancer cell lines. Epithelial-mesenchymal transition (EMT), cell proliferation, migration, and invasion were assessed. EphrinA1-Fc ligand was used to determine the suppressor role of EphA2 receptor-ligand binding. CAF-CM-induced EMT and promoted cancer cell motility even without cell-cell interaction. Treatment with a selective EphA2 inhibitor (ALW-II-41-27) or EphA2-targeted siRNA markedly reduced CAF-CM-induced gastric tumorigenesis. EphrinA1-Fc ligand treatment showing ligand-dependent tumor suppression diminished the EphA2 expression and EMT progression. In contrast, ephrinA1-targeted siRNA did not significantly affect CAF-CM-mediated increases in EphA2 expression and EMT progression. Treatment with VEGF showed effects like CAF-CM in terms of EphA2 activation and EMT progression. CAFs may contribute to gastric tumorigenesis by activating EphA2 signaling pathway in a ligand-independent manner. Our results suggest that ligand-independent activation of EphA2 was triggered by VEGF released from CAF-CM. Our result may partially explain why ligand-dependent tumor suppressor roles of EphA2 are not evident in gastric cancer despite the prominent level of ephrinA1.

Binding of EphrinA5 to RET receptor tyrosine kinase: An in vitro study.

Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cell-cell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.

An Agonistic Antibody to EPHA2 Exhibits Antitumor Effects on Human Melanoma Cells.

EPH receptor A2 (EPHA2) is highly expressed in aggressive types of human cancer, and is expected to be an excellent target molecule for antibody treatments. In this study, we investigated the therapeutic potential of antibody to EPHA2 against melanoma in vitro. We generated three monoclonal antibodies (mAbs) to EPHA2 and examined cell-surface expression by flow cytometry. To investigate the ability to inhibit tumor cell migration therapy with mAbs to EPHA2, we performed a wound scratch assay and invasion assay. We investigated the therapeutic effects of immunotoxins consisting of toxin-conjugated EPHA2 mAbs. All human melanoma cell lines studied expressed EPHA2. Like natural ligand ephrin-A1, one of EPHA2 mAbs, SHM16, inhibited metastatic behavior of cells, such as migration and invasion. In addition, drastic growth inhibition and cytotoxicity were found using immunotoxin-conjugated SHM16. These observations indicate a promising role for EPHA2 as a target in antibody treatments for melanoma, and demonstrate the potential therapeutic effects of an agonistic antibody to EPHA2.