Activated ephrinA3/EphA4 forward signaling induces retinal ganglion cell apoptosis in experimental glaucoma.

Previous studies have demonstrated that EphA4 participates in neuronal injury, and there is a strong interaction between ephrinA3 and EphA4. In this study, we showed that in a rat chronic ocular hypertension (COH) experimental glaucoma model, expression of EphA4 and ephrinA3 proteins was increased in retinal cells, including retinal ganglion cells (RGCs) and Müller cells, which may result in ephrinA3/EphA4 forward signaling activation on RGCs, as evidenced by increased p-EphA4/EphA4 ratio. Intravitreal injection of ephrinA3-Fc, an activator of EphA4, mimicked the effect of COH on p-EphA4/EphA4 and induced an increase in TUNEL-positive signals in normal retinas, which was accompanied by dendritic spine retraction and thinner dendrites in RGCs. Furthermore, Intravitreal injection of ephrinA3-Fc increased the levels of phosphorylated src and GluA2 (p-src and p-GluA2). Co-immunoprecipitation assay demonstrated interactions between EphA4, p-src and GluA2. Intravitreal injection of ephrinA3-Fc reduced the expression of GluA2 proteins on the surface of normal retinal cells, which was prevented by intravitreal injection of PP2, an inhibitor of src-family tyrosine kinases. Pre-injection of PP2 or the Ca2+-permeable GluA2-lacking AMPA receptor inhibitor Naspm significantly and partially reduced the number of TUNEL-positive RGCs in the ephrinA3-Fc-injected and COH retinas. Our results suggest that activated ephrinA3/EphA4 forward signaling promoted GluA2 endocytosis, then resulted in dendritic spine retraction of RGCs, thus contributing to RGC apoptosis in COH rats. Attenuation of the strength of ephrinA/EphA signaling in an appropriate manner may be an effective way for preventing the loss of RGCs in glaucoma.

Interleukin-6 affects pacsin3, ephrinA4 expression and cytoskeletal proteins in differentiating primary skeletal myoblasts through transcriptional and post-transcriptional mechanisms.

Interleukin (IL)-6 is a proinflammatory cytokine released in injured and contracting skeletal muscles. In this study, we examined cellular expression of proteins associated with cytoskeleton organization and cell migration, chosen on the basis of microRNA profiling, in rat primary skeletal muscle cells (RSkMC) treated with IL-6 (1 ng/ml) for 11 days. MiRNA microarray analysis and qRT-PCR revealed increased expression of miR-154-3p and miR-338-3p in muscle cells treated with IL-6. Pacsin3 was downregulated post-transcriptionally by IL-6, but not by IGF-I. Ephrin4A protein was increased both in IL-6- and IGF-I-treated myocytes. IL-6, but not IGF-I, stimulated migratory ability of RSkMC, examined in wound healing assay. Alpha-actinin protein was slightly augmented in RSKMC treated with IL-6, similarly to IGF-I. IL-6, but not IGF-I, upregulated desmin in differentiating RSkMC. IL-6 supplementation caused accumulation of alpha-actinin and desmin in near-nuclear area of muscle cells, which was manifested by increased ratio: mean near-nuclear fluorescence/mean peripheral cytoplasm fluorescence of these proteins. We concluded that IL-6, a known proinflammatory cytokine and a physical activity-associated myokine, acting during differentiation of primary skeletal muscle cells, alters expression of nonmuscle-specific miRNAs. This cytokine causes differential effects on pacsin-3 and ephrinA4, through post-transcriptional inhibition and stimulation, respectively. IL-6-exerted modifications of cytoskeletal proteins in muscle cells include both transcriptional (desmin and dynein heavy chain 5) and post-transcriptional activation (alpha-actinin). Moreover, IL-6 augments near-nuclear distribution of cytoskeletal proteins, alpha-actinin and desmin and promotes migration of myocytes. Such effects suggest that IL-6 plays a role during skeletal muscle regeneration, acting through mechanisms independent of regulation of myogenic program.

Increased EphA/ephrinA expression in hippocampus of pilocarpine treated mouse.

PURPOSE: EphA family receptor tyrosine kinases and their ephrinA ligands are involved in patterning axonal connections during brain development. Although it has been evidenced that these molecules continue to play a key role in synaptic reorganization and plasticity in normal and injured adult brains, their effect still remains unclear during epileptogenesis. Temporal lobe epilepsy (TLE) is the most common form of adult focal epilepsy and often associates with sclerosis of the hippocampus and mossy fiber sprouting (MFS). The purpose of this study is to evaluate the relationship between EphA/ephrinA molecules and epileptogenesis after status epilepticus (SE). METHOD: A mouse model of chronic temporal lobe epilepsy was prepared by intraperitoneal administration of pilocarpine. EphAs/ephrinAs expression levels of the mouse hippocampus areas were detected at different time points after SE by PCR, in situ hybridization and immunohistochemistry. Mossy fiber sprouting was estimated by Neo-Timm staining. RESULT: EphAs/ephrinAs were widely distributed in the hippocampus area. EphA10 and ephrinA4 were increased significantly following epileptogenesis, and mossy fiber sprouting appeared after SE. CONCLUSION: The up-regulation of EphA/ephrinA expression after SE suggests that they are involved in the pilocarpine-induced epileptogenesis.

Ephrin-a4 is involved in retinal neovascularization by regulating the VEGF signaling pathway.

PURPOSE: Retinal neovascularization (NV) is a major cause of blindness. Recent research suggests that factors other than VEGF participate in this process. This study aimed to determine the role of ephrin-A4 in retinal NV. METHODS: The expression and effect of ephrin-A4 was investigated in a mouse model of oxygen-induced retinopathy (OIR) and the RF/6A retina endothelial cell line. Ephrin-A4 expression and VEGF signaling pathway phosphorylation were determined by PCR, immunohistochemistry, and western blot analyses. ShRNA was used to silence ephrin-A4 in vitro and in vivo. Retinal flat mounts and tube formation assays were performed to evaluate ephrin-A4 function in the NV process in vivo and in vitro. RESULTS: Ephrin-A4 was overexpressed in the retina of OIR mice and in RF/6A and RPE cells after CoCl2 stimulation. In vitro, Ephrin-A4/Fc treatment significantly increased the tube number of RF/6A cells on a membrane preparation and the phosphorylation levels of VEGR2, Akt1, and ERK1/2 in RF/6A cells. Moreover, ephrin-A4 knockout markedly suppressed pathologic neovascularization in vivo and inhibited the proliferation and tube formation capacity of RF/6A cells in vitro. Furthermore, in the absence of ephrin-A4, the phosphorylation of VEGFR2, Akt1, and ERK1/2 was defective under VEGF165 stimulation, and the proangiogenic function of VEGF165 was also compromised. CONCLUSIONS: This study suggests that ephrin-A4 plays an important role in retinal NV and is a potential target against retinal NV. The proangiogenic function of ephrin-A4 may be linked to its crucial role in the VEGF signaling pathway.

Ephrin A4 expression in osteosarcoma, impact on prognosis, and patient outcome.

BACKGROUND: Ephrin A4 is one of the ephrin ligand molecules belonging to the tyrosine kinases receptor family. It was originally identified in a T-lymphoma cell line and seen to be expressed in human adult tissue as well as several tumor types. In our previous study, we showed the unique pattern of ephrin A4 immunohistochemical staining, which differed according to the type of examined bone specimens (normal bone, primary, and metastatic osteosarcoma lesions). The aim of the present study is to evaluate the prognostic impact of ephrin A4 expression in a group of primary osteosarcoma patients. MATERIALS AND METHODS: Ephrin A4 immunohistochemical expression was carried out on 47 primary osteosarcoma cases. RESULTS: Ephrin A4 was expressed in 82.9% of osteosarcoma cases with cytoplasmic localization in 58.9% of positive cases. The cytoplasmic pattern was significantly associated with aggressive histopathological types of osteosarcoma (P = 0.02), advanced stage (P = 0.04), the presence of metastasis (P = 0.03), inferior response to neoadjuvent chemotherapy (P = 0.04), and tended to be associated with a shorter event-free survival (P = 0.09). CONCLUSIONS: The cytoplasmic pattern of ephrin A4 could identify a subgroup of primary osteosarcoma patients with a high liability for progression, poor prognosis, and inferior response to chemotherapy.

An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression.

Chronic lymphocytic leukemia (CLL) cell migration into lymphoid tissues is an important aspect of the pathobiology of this disease. Here, we investigated the role of ephrin-A4 (EFNA4) in the transendothelial migration (TEM) capacity of CLL and normal B cells through interacting with endothelial EphA2 (erythropoietin-producing hepatocellular carcinoma). CLL cells showed a remarkable impairment in the adhesion to and transmigration through human umbilical vein endothelial cell (HUVEC) monolayers, correlating with their higher EFNA4 expression. In vitro, TEM was mediated by EFNA4 binding to endothelial EphA2 receptor, which is highly expressed in tumor necrosis factor-alpha-activated HUVECs as well as in the CD31(+) endothelial cells of human lymph nodes. The pretreatment of CLL cells with EphA2 homodimers further impaired their adhesion to and transmigration through HUVEC monolayers, whereas pretreatment of HUVECs with EFNA4 homodimers improved those phenomena in both CLL and normal B cells, suggesting that EFNA4 signaling negatively contributed to TEM. In fact, EFNA4 signaling into CLL cells significantly reduced their adhesion to intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and several extracellular matrix molecules and impaired CCL-19-mediated TEM and chemotaxis. Our results suggest that EFNA4-EphA2 interactions are involved in CLL cell trafficking between blood and the tissues and therefore may become a therapeutic target in the future.

Immunohistochemical profile of ephrin A4 expression in human osteosarcoma.

Ephrin receptors and ephrin ligands constitute one of the largest groups of tyrosine kinases. The division of ephrin receptors into type A or type B is determined by their ligand-binding specificities. Ephrin A4 as a ligand has a broad capacity to bind and stimulate different subtypes of ephrin A receptors. Little is known about the role of ephrins generally and ephrin A4 particularly in osteosarcoma. Ephrin A4 was immunohistochemically assessed on archival material from 46 primary osteosarcoma cases, 10 metastatic pulmonary lesions and 20 normal control bone specimens. Ephrin A4 was expressed in 100% of normal bone specimens, in 84.4% of primary osteosarcoma cases and in all metastatic pulmonary lesions. Cytoplasmic and nucleocytoplasmic patterns of ephrin A4 immunoreactivity were observed, with the predominance of the latter pattern in normal bone (100%), and in 43.5% of primary osteosarcoma cases, which showed a higher intensity of expression compared with normal bone (p<0.05). The cytoplasmic pattern is the only staining pattern seen in metastatic cases, which may suggest its role in enhancement of invasion and metastasis. The differences in the distribution of the two patterns of ephrin A4 may indicate a different biological activity of this molecule depending on its localization. The nuclear localization of ephrin A4 requires further investigation to clarify the mechanism and the significance of the nuclear trafficking of ephrin A4.

Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application.

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.

Ephrin-A5 exerts positive or inhibitory effects on distinct subsets of EphA4-positive motor neurons.

Eph receptor tyrosine kinases and ephrins are required for axon patterning and plasticity in the developing nervous system. Typically, Eph-ephrin interactions promote inhibitory events; for example, prohibiting the entry of neural cells into certain embryonic territories. Here, we show that distinct subsets of motor neurons that express EphA4 respond differently to ephrin-A5. EphA4-positive LMC(l) axons avoid entering ephrin-A5-positive hindlimb mesoderm. In contrast, EphA4-positive MMC(m) axons extend through ephrin-A5-positive rostral half-sclerotome. Blocking EphA4 activation in MMC(m) neurons or expanding the domain of ephrin-A5 expression in the somite results in the aberrant growth of MMC(m) axons into the caudal half-sclerotome. Moreover, premature expression of EphA4 in MMC(m) neurons leads to a portion of their axons growing into novel ephrin-A5-positive territories. Together, these results indicate that EphA4-ephrin-A5 signaling acts in a positive manner to constrain MMC(m) axons to the rostral half-sclerotome. Furthermore, we show that Eph activation localizes to distinct subcellular compartments of LMC(l) and MMC(m) neurons, consistent with distinct EphA4 signaling cascades in these neuronal subpopulations.

Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes.

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.

Human dendritic cells express neuronal Eph receptor tyrosine kinases: role of EphA2 in regulating adhesion to fibronectin.

Eph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+ progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a beta1 integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking.

EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system.

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins-A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon-axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli.