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ALTERATION OF THE EPHA2/EPHRIN-A SIGNALING AXIS IN PSORIATIC EPIDERMIS

ALTERATION OF THE EPHA2/EPHRIN-A SIGNALING AXIS IN PSORIATIC EPIDERMIS

Microarray studies indicate that EphA2 is increased in psoriatic plaques where keratinocyte differentiation is impaired (Jabbari et al., 2012; Kulski et al., 2005; Piruzian et al., 2009). To examine EPH/EFN gene expression in psoriasis in more detail, we first used a previously generated microarray data set to analyze the skin of normal individuals (n=64) compared to paired biopsies obtained from uninvolved and lesional areas of patients with psoriasis (n=58) (Gudjonsson et al., 2010a). A number of changes for these receptors and ligands were revealed when comparing psoriatic plaques (PP) with either non-lesional (PN) or normal (NN) controls (Fig. 2a, Fig. S2 and supplementary Table 1). Of the 14 Eph RTK family members, EPHA2 showed the greatest increase in psoriatic plaques. Interestingly, the other EphA subtypes expressed by keratinocytes (EPHA1, EPHA4) were also elevated in lesional skin. In contrast, the mRNA transcripts for epidermal ephrin-A ligands (EFNA1, EFNA3, EFNA4) were decreased in psoriatic plaques, particularly when compared to uninvolved patient skin. Similar to epidermal ephrin-A ligands, EFNB2 and EFNB3 were reduced in lesional skin. Several EphB family members were also altered in psoriatic plaques, the most striking being an increase in EPHB2 as well as a decrease in EPHB1 and EPHB6; these particular receptor subtypes happen not to be concentrated in keratinocytes (Walsh and Blumenberg, 2011b).
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EphA/ephrin A reverse signaling promotes the migration of cortical interneurons from the medial ganglionic eminence

EphA/ephrin A reverse signaling promotes the migration of cortical interneurons from the medial ganglionic eminence

Inhibitory interneurons control the flow of information and synchronization in the cerebral cortex at the circuit level. During embryonic development, multiple subtypes of cortical interneurons are generated in different regions of the ventral telencephalon, such as the medial and caudal ganglionic eminence (MGE and CGE), as well as the preoptic area (POA). These neurons then migrate over long distances towards their cortical target areas. Diverse families of diffusible and cell-bound signaling molecules, including the Eph/ephrin system, regulate and orchestrate interneuron migration. Ephrin A3 and A5, for instance, are expressed at the borders of the pathway of MGE-derived interneurons and prevent these cells from entering inappropriate regions via EphA4 forward signaling. We found that MGE-derived interneurons, in addition to EphA4, also express ephrin A and B ligands, suggesting Eph/ephrin forward and reverse signaling in the same cell. In vitro and in vivo approaches showed that EphA4-induced reverse signaling in MGE-derived interneurons promotes their migration and that this effect is mediated by ephrin A2 ligands. In EphA4 mutant mice, as well as after ephrin A2 knockdown using in utero electroporation, we found delayed interneuron migration at embryonic stages. Thus, besides functions in guiding MGE-derived interneurons to the cortex through forward signaling, here we describe a novel role of the ephrins in driving these neurons to their target via reverse signaling.
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Thalamic afferents influence cortical progenitors via ephrin A5 EphA4 interactions

Thalamic afferents influence cortical progenitors via ephrin A5 EphA4 interactions

Fig. 5. Evidence for a regulation of cortical progenitors by ephrin A5 expressing thalamic axons. (A-D) Axons of thalamic explants (E14.5+1div) express ephrin A ligands as revealed by EphA3-Fc binding sites. (A) Thalamic explants were treated with EphA3-Fc prior to immunostaining for β III-tubulin (Tubb3). The boxed region is magnified in B-D. (B) Immunostaining against β III-tubulin. (C) EphA3-Fc binding sites visualized with an Alexa 488-labeled anti-Fc antibody. (D) Overlay of B and C. (E-H) Isolated axonal compartments from thalamic explants without soma contamination (E14.5+1div) (E-G; arrowheads indicate isolated axons) contained ephrin A5 transcripts (Efna5, 292 bp fragment) as revealed by RT-PCR (H); the housekeeping gene β -actin (Actb) provided a positive control. Single-cell libraries of preoptic area-derived cells and 3 ′ -enriched cDNA libaries generated from E14.5/E16.5 embryonic brain tissue served as positive controls ( pos. Ctl.); the negative control (neg. Ctl.) lacked cDNA. 1 and 2 refer to the axonal samples. Clean Ctl. and picked Ctl. refer to control conditions in which freshly prepared isolation buffer (clean control) and isolation buffer of the explants after axon isolation ( picked control) were used as templates for cDNA synthesis to check for potential RNA contamination. (I-K) L1 immunostaining in coronal brain sections at E13.5 (I,J) and E14.5 (K). (I,J) At E13.5, L1 signals were detected in the thalamic mantle zone (arrow) and L1-positive fibers were observed growing through the basal telencephalon and entering the cortex (boxed region is magnified in J), which was even more evident at E14.5 (K). (L,M) DiA injections into the thalamus of whole brain cultures revealed DiA-stained fibers entering the cortex (E13.5+10 h). Arrows indicate thalamocortical fibers. (N-Q) Cell Tracker Green-labeled E13.5 single cortical cells co-cultured with either EphA3-Fc-negative (N,O) or EphA3-Fc-positive (P) E13.5 thalamic explants for 24 h and labeled by nestin/ β III-tubulin staining. Cell Tracker Green and clustered EphA3-Fc/Alexa 488 is shown in green. Cell pairs are outlined. (Q) Quantification of the proportion of cell pairs shows an increased proportion of β III-tubulin/ nestin-positive cell pairs (*P<0.05, Student ’ s t-test) in response to EphA3-positive versus EphA3-negative thalamic axons (n refers to cell pairs; four different experiments). Ctx, cortex; Thal, thalamus. Scale bars: 500 µm in I,K; 200 µm in J; 100 µm in A,E-G,L,M; 10 µm in D,N-P. DEVEL
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Presenilin-dependent intramembrane cleavage of ephrin-B1

Presenilin-dependent intramembrane cleavage of ephrin-B1

GPI-anchored type ephrin ligand, is cleaved by ADAM10/ Kuzbanian upon activation by its cognate receptor, and this process is implicated in Eph-ephrin-A mediated axonal repulsion [9]. Our finding that ephrin-B is shed by metalloprotease suggest that Eph-ephrin-B interaction- mediated repulsion might also be regulated by proteoly- sis, in a similar manner to Notch [3]. However, recent reports showed that ephrin-B-mediated repulsion is regu- lated by trans-endocytosis after engagement with EphB receptor [19,20]. Furthermore, we observed that treat- ment of ephrin-B expressing cells with clustered EphB2 fusion protein had no effect on shedding as well as γ- secretase-mediated cleavage of ephrin-B (data not shown). We also found that ephrin-B stub undergoes γ- secretase-dependent intramembrane cleavage independ- ently of its cytoplasmic C terminus, that contains a PDZ domain binding motif and conserved tyrosines. These findings suggest that γ-secretase cleavage of ephrin-B is not regulated by a protein-protein interaction within its C-terminal region. These data also indicate that the prote- olytic processing of ephrin-B represents a constitutive sig- naling or metabolic pathway, that may be independent of the interaction with EphB2 receptor in COS cells. How- ever, it has been shown that ephrin-B can interact with other EphB receptors including EphA4. Further character- ization using a complete set of Eph receptors and/or cell lines is needed for the elucidation of the precise role of proteolytic processing of ephrin-B in its signaling. To date, functions of γ-secretase-generated ICD as tran- scriptional activator (i.e., Notch, APP, CD44) or repressor (i.e., Jagged, N-cadherin) within nucleus have been reported [3]. We found that proteolytically generated eB1ICD, that is highly labile, localizes to the nucleus. Moreover, deletion of the acidic amino acid stretch located at the cytosolic face diminished the nuclear local- ization of this intracellular fragment, inferring its role as a nuclear transcriptional regulator. Although eB1ICD lacks known transactivation domain, intracellular domains of a subset of γ-secretase substrates interact with co-transcrip- tional activators after cleavage to facilitate nuclear translo- cation and/or transcriptional regulation (i.e, RBPJk for Notch [21], Fe65 for APP [22], YAP for ErbB-4 [23]). Fur- ther proteomic and genetic approach will be needed to clarify if eB1ICD regulates gene transcription.
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Ephrin-A2 and ephrin-A5 guide contralateral targeting but not topographic mapping of ventral cochlear nucleus axons

Ephrin-A2 and ephrin-A5 guide contralateral targeting but not topographic mapping of ventral cochlear nucleus axons

Role of ephrin-A2 and ephrin-A5 in topographic targeting Topographic mapping is seen throughout the central nervous system and represents a fundamental organizing principle of sensory pathways. Topography in a number of pathways has been shown to arise from graded expression of Eph proteins together with varying degrees of activity-dependent refinement [30–32]. In visual system pathways, formation of retinotopy relies exten- sively on gradients of ephrin-A proteins [33, 34]. In con- trast, we found that null mutations in ephrin-A2 and ephrin-A5 had no effect on topographic mapping in the VCN-MNTB pathway. Previous studies have suggested that EphB proteins are needed for formation of tonotopy in MNTB [16], the inferior colliculus [32, 35], and the auditory cortex [36]. Our results suggest that ephrin-A2 and ephrin-A5 are not predominant factors in establish- ment of the VCN-MNTB map in the brainstem. The role of these proteins has not been established in projec- tions from MNTB to its targets, including LSO. The involvement of ephrin-A proteins is not ruled out in other tonotopic projections, as EphA7 modulates tono- topy in the corticothalamic projection from auditory cortex [37]. Moreover, ephrin-A2/A5 DKO mice show impaired topography in an experimentally induced ret- inal projection into the medial geniculate nucleus of the thalamus, which normally receives auditory input [38]. These observations suggest a role for EphA signaling in the formation of topography in the auditory thalamus. The roles of other ephrin-A proteins in establishing tonotopy have yet to be identified.
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Roles of EphB3/ephrin B1 in feather morphogenesis

Roles of EphB3/ephrin B1 in feather morphogenesis

Following the stabilization of feather primordia, a new epidermal domain is generated between the bud and interbud domain. This new domain invaginates into the dermis, leading to the formation of a feather follicle, a critical property of skin appendages (Chuong and Homberger, 2003; Maderson, 2004; Jiang et al., 2011). Subse- TXHQWO\WKHDSSDUHQWO\KRPRJHQRXVIHDWKHUÀODPHQWF\OLQGHUVWDUWV to generate periodically arranged barb ridges (Prum, 1999; Harris et al., 2002; Yu et al., 2002; Chuong, 2003). They form alternatively arranged growth and apoptotic epidermal domains, leading to the formation of feather branches with intervening space (Chang et al., 2004b). Thus, the epidermis is transformed from a two-dimensional sheet into a complex three-dimensional structure. During this process, new domains emerge, become established, and take on different differentiation fates (Chang et al., 2004a; Alibardi and Toni, 2008; Alibardi, 2010a; Alibardi, 2010b). Failure to segregate these domains leads to inter-mixing of cell types and improper morphogenesis. While we have learned that molecules such as FGFs, BMPs and Wnts (Noramly and Morgan, 1998; Noramly et al., 1999; Widelitz et al., 2000; Harris et al., 2004; Jiang et al., 2004) are involved in the initiation of feather buds, and Shh is involved in subsequent feather growth (Ting-Berreth and Chuong, 1996a; Yu et al., 2002), we have not learned much about the molecules involved in the segregation of tissue primordia from one another during feather morphogenesis, so-called boundary establishment. In recent years, the Eph receptor tyrosine kinases and their ephrin ligands have garnered increasing attention due to their dy- namic properties. Ephrin ligands and their receptors, Ephs, are cell membrane molecules now widely known to be involved in cell-cell interactions through cell adhesion and repulsion (Patan, 2004). Eph ZDVÀUVWLGHQWLÀHGLQDQHU\WKURSRLHWLQSURGXFLQJKHSDWRFHOOXODU carcinoma cell line (Hirai et al., 1987) and belongs to the receptor tyrosine kinase family (Pasquale, 2005). The Eph receptors elicit forward signals and ephrins provide reverse signals (Davy et al., 2004). There are 16 known receptors with 14 found in mammals (Pitulescu and Adams, 2010). As a rule EphA receptors bind to ephrin-A ligands, which are anchored to the membrane through glycosylphosphatidylinositol (GPI) linkage. EphB receptors bind to the transmembrane ephrin-B ligands, (Pasquale, 2005). However, EphA4 receptors also can bind to ephrin-Bs and EphB2 receptors can also bind to ephrin-A5 (Pasquale, 2010). The formation of Eph tetramers is necessary to elicit biological activity (Vearing et al., 2005). Signaling complexity is derived from the composition and signal capabilities of homo- and heterotypic ephrin-Eph clusters (Janes et al., 2012).
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Eph/ephrin signalling during development

Eph/ephrin signalling during development

Migration of cells or cellular processes on top of another cell substrate requires a fine balance of attractive/adhesive and repulsive forces to allow migration while preventing migrating cells from invading the underlying tissue. During Xenopus gastrulation, migration of the mesoderm over the ectoderm requires multiple EphB and ephrin B proteins on each side of the boundary. The migrating mesodermal cells alternate between attachment mediated by (unknown) adhesion proteins and detachment triggered by EphB forward signalling (Rohani et al., 2011). In the peripheral nervous system, ephrin A-positive sensory axons track along EphA-positive motor axons. This tracking event is independent of EphA forward signalling but requires that the EphA ectodomain triggers ephrin A-mediated attraction/adhesion. Loss of motor EphA3/4 or sensory ephrin As shifts the balance towards repulsion, mediated by an as yet unidentified activity (Wang et al., 2011).
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The role of the Eph ephrin signalling system in the regulation of developmental patterning

The role of the Eph ephrin signalling system in the regulation of developmental patterning

Analysis of Eph and ephrin protein-protein interactions show that many of the receptors show cross-reactivity which is usually restricted to either GPI-linked or transmembrane ephrins (Gale et al., 1996; Flanagan and Vanderhaeghen, 1998). These observa- tions led to the classification of receptors into A or B categories depending on their preference for binding of GPI-linked or trans- membrane ephrins respectively (Lemke, 1997). This early notion that a single Eph protein could bind with similar affinity to all ephrins of a certain class has proven to be inaccurate. Indeed, some interactions are relatively specific, for example EphB4 binds strongly to ephrin B2 but weakly to other ephrins (Gerety et al., 1999). At the other extreme EphA4 binds members in both ephrin A and ephrin B subgroups with comparable affinities (Gale et al., 1996) and, as discussed below, probably binds the ephrin B family in many of its most critical developmental roles. Even those receptors, which appear cross-reactive, exhibit an ordering in affinities of interaction with preferred high affinity ephrin interactions. An example is EphA3 which binds several A ephrins and even shows weak binding to B ephrins but binds ephrin A5 with a much higher affinity than other ephrins (Lackmann et al., 1997). Individual Eph-ephrin interactions have been shown to have a strict one to one stoichiom- etry (Lackmann et al., 1997). The binding affinity is principally determined by the rate of dissociation and thus critically deter- mines the average half life of a particular Eph/ephrin complex. It follows that this in turn determines the probability of oligomerization of Eph-ephrin complexes, an essential requirement for triggering of autophosphorylation and signal transduction (Stein et al., 1998).
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Exploring the Horizons of Ephrin B2 Receptor for Combating Paramoxyviridae Infection

Exploring the Horizons of Ephrin B2 Receptor for Combating Paramoxyviridae Infection

The Hendra and Nipah viruses (HeV and NiV) are prominant members of the Paramyxoviridae family [1]. The initial cases were detected in Australia in 1994-95 and Malaysia in 1998-99. Henipa viruses have been transmitted to humans through several sources [2]. Henipa virus interactions with target cells are mediated by its 75 kDa glycoprotein moiety attachment and 70 kDa fusion proteins attachment respectively, where both of them are evident prospects for membrane fusion. Recently Ephrin-B2 has

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The effects of olanzapine on genome-wide DNA methylation in the hippocampus and cerebellum

The effects of olanzapine on genome-wide DNA methylation in the hippocampus and cerebellum

pathways. A list of 123 such genes that increased in methylation in both brain regions is given in Additional file 9: Table S5. That methylation may serve an inter- mediary role in modulating gene expression is apparent in the cerebellum, which is dominated by a number of signalling pathways including ephrin receptors and syn- aptic long-term potentiation (Table 2). Ephrin ligands and receptors guide axons during neural development and regulate neuronal plasticity in adults [55,56]. Specif- ically, ephrin plays an important role in the regulation of neuronal migration, which is essential for the develop- ment of the nervous system and the proper functioning of the brain [57]. Neuronal cells have ahigher variation in DNA methylation than non-neuronal cells, supporting the idea that the epigenetic status of neuronal cells changes in response to the environment in the brain
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Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer

Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer

The purpose of using ephrin-A1–LNP to deliver miR is to enhance the transfection efficiency on lung cancer cells by cell surface receptor EphA2 targeted delivery. In addition, our study provides ephrin-A1 and let-7a miR combination therapy for MPM and NSCLC. The miR–ephrin-A1–LNP complex is highly effective in inhibiting cell proliferation, cell migration, and tumor growth compared to ephrin-A1– LNP and miR–LNP. In addition, the enhanced inhibitory effect of miR–ephrin-A1–LNP implies that the let-7a miR were successfully transfected in the lung cancer cells via the ephrin-A1–LNP delivery. Furthermore, in the wound healing assay, the miR–ephrin-A1–LNP resulted in greater reduction on cell migration rate than did miR–LNP and ephrin-A1–LNP treatments. Although the positive charge on LNP surface was attenuated by ephrin-A1 conjugation, which usually results in slower diffusion of liposomes into the cell membrane, the cellular uptake of ephrin-A1–LNP was enhanced by the specific receptor-ligand affinity of ephrin-A1 and EphA2 expressing cancer cells. This suggests that the combination treatment of ephrin-A1 and let-7a miR was highly effective
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Temporal regulation of ephrin/Eph signalling is required for the spatial patterning of the mammalian striatum

Temporal regulation of ephrin/Eph signalling is required for the spatial patterning of the mammalian striatum

Several mouse mutants have been reported to display alterations in the specification of matrix or striosome neurons, including Mash1 ( Ascl1 ), Notch1 and CTIP2 ( Bcl11b ) mutant mice, where early striatal populations are reduced in size or lost (Arlotta et al., 2008; Casarosa et al., 1999; Mason et al., 2005), and Dlx1 / 2 and Ebf1 mutants, where the matrix population is mostly affected (Anderson et al., 1997; Garel et al., 1999). However, these mutants display no defect in striatal compartmentalization per se, so that EphA4 mutants constitute the first model of selective disruption of the cytoarchitecture of the striatum. Matrix/striosome organization has been shown to be important for several aspects of striatal function, but the exact relationships between striatal function and cytoarchitecture remain poorly known. Ephrin A5/EphA4 mutants constitute the first example of genetic disruption of the cytoarchitecture of the striatum, and thereby constitute a unique model with which to test how the disruption of matrix/striosome compartments might be associated with abnormalities in connectivity and function of the basal ganglia, and how these may be related to abnormal behavioural traits.
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Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord

Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord

A survey of members of the B-type Eph receptor family in the mouse ventral spinal cord (Fig. 1a) indicated that spinal progenitors co-express several EphB receptors, as well as EphA4, as shown by in situ hybridization (Fig. 1b-d) and immunofluorescence (Fig. 1e-g). Concerning B-type ephrin ligands, in situ hybridization at different developmental stages reveals that while Efnb1 is not expressed at significant levels in progenitors of the ventral spinal cord (Fig. 1h-j), both Efnb2 and Efnb3 are expressed in subsets of these cells. More precisely, at all stages analyzed, Efnb2 is expressed by progenitors located at an intermediate dorso- ventral position within the spinal cord, its expression never extending to the ventral-most region (Fig. 1k-m). Con- versely, expression of Efnb3 is highest in the ventral-most region of the spinal cord at all stages analyzed, with a lower expression extending more dorsally (Fig. 1n-p). Because Efnb2 and Efnb3 were expressed in distinct progenitor domains of the spinal cord, we asked whether these corre- sponded to progenitors with distinct identities, namely pMN progenitors expressing Olig2 and p3 progenitors expressing Nkx2.2. Since the expression of Efnb2 in pro- genitors of the ventral neural tube detected by in situ hybridization was low, we took advantage of a reporter mouse line that expresses H2BGFP under the control of the Efnb2 endogenous promoter [22]. The benefit of this reporter strategy is that H2BGFP accumulates in the nu- cleus thus highlighting low domains of expression and fa- cilitating co-expression analyses. In accordance with in situ hybridization data, H2BGFP expression was detected in a restricted population of neural progenitors from E9.5 to E11.5 (Fig. 2a-d). Co-staining with Olig2 showed that the expression domain of Efnb2 overlapped with the Olig2 + (pMN) domain (Fig. 2a-l). Co-staining with Olig2 and Nkx2.2, the iTFs for pMN and p3 respectively, showed that the ventral boundary of Efnb2 expression strictly corre- sponds to the p3/pMN boundary (Fig. 2m-p). Conversely, in situ hybridization for Efnb3 followed by Olig2 immuno- staining showed that the highest domain of Efnb3 expres- sion corresponds to Olig2 - floor plate and p3 progenitors (Fig. 2q-s). Altogether, these expression analyses indicate that all progenitors of the ventral spinal cord co-express several Eph receptors and reveal that ephrinB2 and
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Outstanding questions in developmental ERK signaling

Outstanding questions in developmental ERK signaling

For example, during the induction of otx in the Ciona embryo, the effect of FGF is spatially restrained by the Eph/ephrin pathway another juxtracrine signaling system that provides negat[r]

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Identification and characterization of endothelial specific genes

Identification and characterization of endothelial specific genes

The detailed analysis of Depp-Cre activity and ephrin-B2 expression in the heart reveals that Depp-Cre mediated recombination occurs in a subset of endocardial and myocardial cells of the outflow tract (Fig. 3D-F), and ephrin-B2 is expressed in the endocardial and myocardial cells of the outflow tract but barely expressed in myocardial cells of the atria and ventricles (Fig. 3A-C, and data not shown). Therefore, it is not clear whether the severe heart defects and the angiogenesis defects witnessed in the half of the conditional mutants at E9.5 are caused by ephrin-B2 deletion in peripheral endothelial cells or by the deletion in the endocardial and/or myocardial cells of the outflow tract.
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Proper closure of the optic fissure requires ephrin A5 EphB2 JNK signaling

Proper closure of the optic fissure requires ephrin A5 EphB2 JNK signaling

To identify the receptor for ephrin A5 in the proximoventral optic cup at E11.5, we visualized the binding of EphA8 and ephrin A5 Fc fusion proteins. As expected, EphA8-Fc binds primarily to the nasal retina because of its high expression of ephrin A5 (Fig. 2A,B). Ephrin A5-Fc, however, binds primarily to the proximal optic cup (Fig. 2C, Fig. S2A,B), where we consistently observed apoptotic cell death. EphA mRNAs were barely detectable via RNA in situ hybridization along the proximoventral optic cup at E11.5 (data not shown). In addition, we also observed ephrin B1-Fc binding to the proximoventral optic cup (Fig. 2D), suggesting that EphB genes are potential receptors for ephrin A5. This would provide in vivo physiological relevance to the published finding that ephrin A5 is capable of binding to and activating the EphB2 receptor (Himanen et al., 2004). Consistent with this hypothesis, we observed binding of ephrin A5-Fc to both EphA8 and EphB2 in HEK293 cells (Fig. 2E-G), whereas ephrin B1-Fc is only capable of binding to EphB2 (Fig. 2H-J). In addition to EphB2, ephrin A5-Fc also binds EphB1 but not EphB3 (Fig. 2K-R). Together, these results suggest either EphB1 or EphB2 as the physiologically relevant receptor for ephrin A5 in the proximoventral optic cup at E11.5.
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Soluble ephrin a1 is necessary for the growth of HeLa and SK-BR3 cells

Soluble ephrin a1 is necessary for the growth of HeLa and SK-BR3 cells

The localization of EPHA2 away from cell-cell con- tacts is correlated with its transformation properties [16,17]. Our results show that soluble EFNA1 is impor- tant for this relocalization in HeLa cells. In cells deficient for EFNA1 or over-expressing full length mem- brane bound EFNA1, EPHA2 returns to sites of cell-cell contact and is no longer transforming. Whereas in cells overexpressing soluble EFNA1, the localization of EPHA2 resembles that of wild-type HeLa cells, and is not enriched at cell-cell contacts. These results suggest that soluble EFNA1 is involved in the relocalization of EPHA2 during transformation. One possibility is that soluble EFNA1 competes for binding of other ephrins to EPHA2 at cell-cell contacts and thereby draws EPHA2 away from cell contacts. If membrane bound EFNA1 is important for anchoring EPHA2 at cell-cell contacts and soluble EFNA1 relocalizes EPHA2 away from these sites, what causes EPHA2 to return to cell-cell contacts in EFNA1 knockdown cells? We have found that HeLa cells also express ephrin A2 and A4 (data not shown). Therefore, EPHA2 localization may be restored through an interaction with these ephrins. However, further work is required to determine how soluble EFNA1 con- tributes to EPHA2 localization and signaling, and to test these possibilities.
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Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

We initially considered efnA5 localized in motor neuron neighbouring cells such as astrocytes as a possible binding ligand of EphA4. However, we observed predominant neuronal efnA5 expression. Reducing efnA5 expression with 50% accelerated disease progression, whereas hetero- zygous deletion of EphA4 gene increased survival in SOD1 G93A mice [42]. Although highly speculative efnA5 and EphA4 expressed in the same motor neurons could establish inhibitory cis-binding. The absence of efnA5 may increase EphA4 signalling, resulting in shorter survival in mice. Inhibitory cis-binding has already been reported for efnA5 in retinal axons, where cis-binding of efnA5 with EphA3 desensitizes EphA3-mediated signalling [7]. Future research could be focussed on further elucidating this hypothesis and considering the implications for other ephrin members of the family. In addition, efnA5 is a ligand of most EphA receptors [30]. Since the interaction of Ephs and ephrins mediate intercellular communication [20, 34], an- other hypothesis would be that efnA5 mediates neuropro- tection by binding in trans to other EphA receptors expressed in other cell types surrounding neurons. Further work will address whether these other Eph receptors might have a role in ALS. Interestingly, a neuroprotective response has been suggested for the neuronal EphB1, which interacts with the astrocytic ephrin-B1 to induce a neuroprotective phenotype in astrocytes [41].
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Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification

Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification

To determine how chondrocytes support cartilage destruction, we focused on two possible activities: the support of osteoclast formation by Efnb2- deficient chondrocytes, and their expression of cartilage-degrading enzymes. Co-culture of differentiated primary chondrocytes from Osx1Cre.Efnb2 Δ/Δ mice showed impaired support of osteoclast formation, as we previously observed with osteoblasts derived from mice of the same genotype (Tonna et al., 2014). This was also consistent with the work of others showing that specific inhibition of the ephrin B2- EPHB4 interaction with the TNYL-RAW peptide inhibited osteoclast formation supported by the ATDC5 chondrocyte cell line (Wang et al., 2014). Surprisingly, mRNA levels for RANKL, a ligand that supports osteoclast formation and is expressed by hypertrophic chondrocytes (Kartsogiannis et al., 1999), and for the RANKL decoy receptor OPG were unchanged. This supports a model whereby the ephrin B2/EPHB4 role in chondrocytic support of osteoclastogenesis is independent of the RANKL/OPG system, as previously suggested (Wang et al., 2014). We hypothesized that enzymatic degradation of the cartilage matrix may also play a role in the osteoclastic destruction of cartilage surrounding hypertrophic chondrocytes.
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Eph/ephrin interactions modulate muscle satellite cell motility and patterning

Eph/ephrin interactions modulate muscle satellite cell motility and patterning

Interestingly, we also observed oriented differentiation in vitro in response to ephrin stripes. Labeled satellite cells plated on Alexa fluor control stripes or ephrin-B1 stripes were incubated for 24 hours then switched into low serum media without FGF2 and incubated for an additional 48 hours to promote satellite cell differentiation. Cells plated on control stripes differentiated in small radial clusters, as is usually seen in vitro, whereas on ephrin-B1- programmed coverslips, the satellite cells were primarily located on the ephrin-free areas (as would be expected from the motility studies) and were aligned and differentiated in parallel to the Fc:ephrin stripes (Fig. 4A). However, when plated on stripes of ephrin-A1 (which did not affect satellite cell motility in vitro), no parallel alignment of differentiated cells was observed (Fig. 4A). Differentiating satellite cells did not express EphA1, the only Eph receptor specific for ephrin-A1 (Fig. 4B), but they robustly expressed EphA2 (Fig. 4B), which is the primary signaling receptor for ephrin-A1 in many systems (for a review, see Pasquale, 2010). EphA2 binds and signals in response to ephrin-A2, ephrin-A3 and ephrin-A5, and oriented, parallel myotube patterning was observed in response to stripes programmed with Fc:ephrin chimeras of each (Fig. 4B,C). These data suggest that although patterning of nascent myotubes is affected by specific Eph/ephrin interactions, the actual signaling complex might be more complicated than a simple one- to-one correlation of an Eph and an ephrin. In particular, crosstalk from other adhesion receptors, which has been observed in many other systems (for a review, see Arvanitis and Davy, 2008), might influence satellite cell responses to ephrin stimulation. Future work will focus on defining potential Eph-ephrin pair(s) involved in myotube patterning, the stage(s) of differentiation at which Eph/ephrin signaling impinges on myotube patterning, and other signaling pathways that might also be acting to mediate myotube alignment in response to ephrin.
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