EphA7 regulates claudin6 and pronephros development in Xenopus

Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph receptor EphA7 and its soluble form in Xenopus pronephros development. EphA7 is specifically expressed in pronephric tubules at tadpole stages and knockdown of EphA7 by a translation blocking morpholino led to defects in tubule cell differentiation and morphogenesis. A soluble form of EphA7 (sEphA7) was also identified. Interestingly, the membrane level of claudin6 (CLDN6), a tetraspan transmembrane tight junction protein, was dramatically reduced in the translation blocking morpholino injected embryos, but not when a splicing morpholino was used, which blocks only the full length EphA7. In cultured cells, EphA7 binds and phosphorylates CLDN6, and reduces its distribution at the cell surface. Our work suggests a role of EphA7 in the regulation of cell adhesion during pronephros development, whereas sEphA7 works as an antagonist.     

EphA7 regulates spiral ganglion innervation of cochlear hair cells

During the development of periphery auditory circuitry, spiral ganglion neurons (SGNs) form a spatially precise pattern of innervation of cochlear hair cells (HCs), which is an essential structural foundation for central auditory processing. However, molecular mechanisms underlying the developmental formation of this precise innervation pattern remain not well understood. Here, we specifically examined the involvement of Eph family members in cochlear development. By performing RNA‐sequencing for different types of cochlear cell, in situ hybridization, and immunohistochemistry, we found that EphA7 was strongly expressed in a large subset of SGNs. In EphA7 deletion mice, there was a reduction in the number of inner radial bundles originating from SGNs and projecting to HCs as well as in the number of ribbon synapses on inner hair cells (IHCs), as compared with wild‐type or heterozygous mutant mice, attributable to fewer type I afferent fibers. The overall activity of the auditory nerve in EphA7 deletion mice was also reduced, although there was no significant change in the hearing intensity threshold. In vitro analysis further suggested that the reduced innervation of HCs by SGNs could be attributed to a role of EphA7 in regulating outgrowth of SGN neurites as knocking down EphA7 in SGNs resulted in diminished SGN fibers. In addition, suppressing the activity of ERK1/2, a potential downstream target of EphA7 signaling, either with specific inhibitors in cultured explants or by knocking out Prkg1, also resulted in reduced SGN fibers. Together, our results suggest that EphA7 plays an important role in the developmental formation of cochlear innervation pattern through controlling SGN fiber ontogeny. Such regulation may contribute to the salience level of auditory signals presented to the central auditory system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 452–469, 2016     

Dissociation of corticothalamic and thalamocortical axon targeting by an EphA7-mediated mechanism

Molecular mechanisms generating the topographic organization of corticothalamic (CT) circuits, which comprise more than three-quarters of the synaptic inputs onto sensory relay neurons, and their interdependence with thalamocortical (TC) axon development are unknown. Using in utero electroporation-mediated gene transfer, we show that EphA7-mediated signaling on neocortical axons controls the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that mis-express EphA7 do not shift the relative positioning of their pathway within the subcortical telencephalon (ST), indicating that they do not depend upon EphA7/ephrin-A signaling in the ST for establishing this topography. Moreover, mis-expression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. Our results support a model in which EphA/ephrin-A signaling controls independently the precision with which CT and TC projections develop, yet is essential for establishing their topographic reciprocity.     

Opposing gradients of ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system

During development of the retinocollicular projection in mouse, retinal axons initially overshoot their future termination zones (TZs) in the superior colliculus (SC). The formation of TZs is initiated by interstitial branching at topographically appropriate positions. Ephrin-As are expressed in a decreasing posterior-to-anterior gradient in the SC, and they suppress branching posterior to future TZs. Here we investigate the role of an EphA7 gradient in the SC, which has the reverse orientation to the ephrin-A gradient. We find that in EphA7 mutant mice the retinocollicular map is disrupted, with nasal and temporal axons forming additional or extended TZs, respectively. In vitro, retinal axons are repelled from growing on EphA7-containing stripes. Our data support the idea that EphA7 is involved in suppressing branching anterior to future TZs. These findings suggest that opposing ephrin-A and EphA gradients are required for the proper development of the retinocollicular projection.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

Conformational Clamping by a Membrane Ligand Activates the EphA2 Receptor

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression.     

EphA Receptors Form a Complex with Caspase-8 to Induce Apoptotic Cell Death

EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 co-precipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinase-inactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptor-like protein acts as a biochemical linker between the Eph receptor and caspase-8.     

Association of EphA4 polymorphism with swine reproductive traits and mRNA expression of EphA4 during embryo implantation

The swine erythropoietin-producing hepatocellular A4 (EphA4) gene, which was detected in the endometrium during embryo implantation in pigs, was one of the potential candidate genes for reproductive traits. In the study, two single nucleotide polymorphism (SNP) loci (EphA4_1 and EphA4_2) in exon 3 of EphA4 gene were analyzed to determine whether EphA4 influenced total number born (TNB) and number born alive (NBA). Association of two diallelic polymorphisms with reproductive traits was assessed in Landrace, Yorkshire and Duroc populations with 2,014 litter records of 765 sows. The results showed that G allele at EphA4_1 locus or C allele at EphA4_2 locus seemed to have advantageous effects on litter size. And the combined analyzed results demonstrated that genotype AGTC, AGCC and GGCC are better than genotype AATT, AATC and AGTT for TNB and NBA in either single parity or all parities. The results in this study demonstrated that EphA4 gene was significantly associated with litter size in pigs. In addition, a high mRNA expression of EphA4 was found in small intestine, large intestine, stomach and endometrium, and the expression decreased during implantation in pigs. Further studies were needed to confirm these preliminary researches.     

4-Substituted quinazoline derivatives as novel EphA2 receptor tyrosine kinase inhibitors

Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2.     

MicroRNA-335 suppresses the proliferation,migration, and invasion of breast cancer cells by targeting EphA4

MicroRNAs (miRNAs) are small noncoding RNAs that exert their functions by targeting specific mRNA sequences. Many studies have demonstrated that miRNAs are crucial for cancer progression, during which they can act as either oncogenes or tumor suppressors. Previous research has shown that miR-335 is downregulated in breast cancer, and it has been shown to be a breast cancer suppressor. In addition, emerging evidence indicates that erythropoietin-producing hepatocellular A4 (EphA4) is implicated in cancer cell proliferation, migration, and invasion. However, little is known about the relationship between miR-335 and EphA4 in breast cancer. In the present study, we used bioinformatic and biochemical analyses to demonstrate that EphA4 is a direct downstream target of miR-335 in human breast cancer MCF-7 and MDA-MB-23 cells and revealed that miR-335 negatively regulates the expression of EphA4 in these cells. Further investigation revealed that miR-335 overexpression inhibits MCF-7 and MDA-MB-231 cell proliferation and that this inhibition is attenuated by EphA4 coexpression. Similarly, miR-335 overexpression also inhibited growth and downregulated EphA4 expression in tumors in nude mice. Moreover, our results demonstrated that miR-335 overexpression suppresses migration and invasion in MCF-7 and MDA-MB-231 cells, an effect that was reversed by EphA4 overexpression. These findings confirmed that EphA4 is a direct target gene of miR-335 and that miR-335 suppresses breast cancer cell proliferation and motility in part by directly inhibiting EphA4 expression.     

EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia

Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.