Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

Endothelial deletion of EPH receptor A4 alters single-cell profile and Tie2/Akap12 signaling to preserve blood-brain barrier integrity.

Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice promotes blood-brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.

EphA4 Induces the Phosphorylation of an Intracellular Adaptor Protein Dab1 via Src Family Kinases.

Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.

Effects of lncRNA HOXA11-AS on Sevoflurane-Induced Neuronal Apoptosis and Inflammatory Responses by Regulating miR-98-5p/EphA4.

Objective: To explore the molecular mechanism of sevoflurane-induced neurotoxicity and to determine whether lncRNA HOXA11-AS affects sevoflurane-induced neuronal apoptosis and inflammation by regulating miR-98-5p/EphA4. Methods: Morris water maze (MWM) test was used to detect the learning and memory ability of rats, HE staining was used to observe hippocampal pathology, TUNEL staining was used to detect the level of neuronal apoptosis, and RT-qPCR was used to detect the expression of HOXA11-AS, miR-98-5p, IL-6, IL-1ß, and TNF-α. At the same time, the contents of IL-6, IL-1ß, and TNF-α in serum were detected by ELISA. The expressions of apoptosis-related proteins EphA4, Bax, Cleaved caspase 3, and Bcl-2 were detected by Western blot. The dual-luciferase gene reporter verified the targeting relationship between HOXA11-AS and miR-98-5p and the targeting relationship between miR-98-5p and EphA4. Results: The expression of HOXA11-AS was observed in sevoflurane-treated rats or cells and promoted neuronal apoptosis and inflammation. HOXA11-AS was knocked out alone, or miR-98-5p was overexpressed which attenuates neuronal apoptosis and inflammatory inflammation after sevoflurane treatment. Furthermore, knockdown of HOXA11-AS alone was partially restored by knockdown of miR-98-5p or overexpression of EphA4. Conclusion: Inhibition of lncRNA HOXA11-AS attenuates sevoflurane-induced neuronal apoptosis and inflammatory responses via miR-98-5p/EphA4.

A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain.

The Eph receptor tyrosine kinases and their ephrin ligands regulate many physiological and pathological processes. EphA4 plays important roles in nervous system development and adult homeostasis, while aberrant EphA4 signaling has been implicated in neurodegeneration. EphA4 may also affect cancer malignancy, but the regulation and effects of EphA4 signaling in cancer are poorly understood. A correlation between decreased patient survival and high EphA4 mRNA expression in melanoma tumors that also highly express ephrinA ligands suggests that enhanced EphA4 signaling may contribute to melanoma progression. A search for EphA4 gain-of-function mutations in melanoma uncovered a mutation of the highly conserved leucine 920 in the EphA4 sterile alpha motif (SAM) domain. We found that mutation of L920 to phenylalanine (L920F) potentiates EphA4 autophosphorylation and signaling, making it the first documented EphA4 cancer mutation that increases kinase activity. Quantitative Föster resonance energy transfer and fluorescence intensity fluctuation (FIF) analyses revealed that the L920F mutation induces a switch in EphA4 oligomer size, from a dimer to a trimer. We propose this switch in oligomer size as a novel mechanism underlying EphA4-linked tumorigenesis. Molecular dynamics simulations suggest that the L920F mutation alters EphA4 SAM domain conformation, leading to the formation of EphA4 trimers that assemble through two aberrant SAM domain interfaces. Accordingly, EphA4 wild-type and the L920F mutant are affected differently by the SAM domain and are differentially regulated by ephrin ligand stimulation. The increased EphA4 activation induced by the L920F mutation, through the novel mechanism we uncovered, supports a functional role for EphA4 in promoting pathogenesis.

Myelination defects in the medial prefrontal cortex of Fkbp5 knockout mice.

The hypothalamic-pituitary-adrenal (HPA) axis plays a principal role in stress response regulation and has been implicated in the etiology of stress-related disorders. The HPA axis regulates the normal synthesis and release of glucocorticoids; dysregulation of the HPA axis causes abnormal responses to stress. FK506-binding protein 5 (FKBP5), a co-chaperone of heat shock protein 90 in the glucocorticoid receptor (GR) molecular complex, is a key GR sensitivity regulator. FKBP5 single nucleotide polymorphisms are associated with dysregulated HPA axis and increased risk of stress-related disorders, including posttraumatic stress disorder (PTSD) and depression. In this study, we profiled the microRNAs (miRNAs) in the medial prefrontal cortex of Fkbp5 knockout (Fkbp5-/- ) mice and identified the target genes of differentially expressed miRNAs using sequence-based miRNA target prediction. Gene ontology analysis revealed that the differentially expressed miRNAs were involved in nervous system development, regulation of cell migration, and intracellular signal transduction. The validation of the expression of predicted target genes using quantitative polymerase chain reaction revealed that the expression of axon development-related genes, specifically actin-binding LIM protein 1 (Ablim1), lemur tyrosine kinase 2 (Lmtk2), kinesin family member 5c (Kif5c), neurofascin (Nfasc), and ephrin type-A receptor 4 (Epha4), was significantly decreased, while that of brain-derived neurotrophic factor (Bdnf) was significantly increased in the brain of Fkbp5-/- mice. These results suggest that axonal development-related genes can serve as potential targets in future studies focused on understanding the pathophysiology of PTSD.

EphA4 Is Required for Neural Circuits Controlling Skilled Reaching.

Skilled forelimb movements are initiated by feedforward motor commands conveyed by supraspinal motor pathways. The accuracy of reaching and grasping relies on internal feedback pathways that update ongoing motor commands. In mice lacking the axon guidance molecule EphA4, axonal misrouting of the corticospinal tract and spinal interneurons is manifested, leading to a hopping gait in hindlimbs. Moreover, mice with a conditional forebrain deletion of EphA4, display forelimb hopping in adaptive locomotion and exploratory reaching movements. However, it remains unclear how loss of EphA4 signaling disrupts function of forelimb motor circuit and skilled reaching and grasping movements. Here we investigated how neural circuits controlling skilled reaching were affected by the loss of EphA4. Both male and female C57BL/6 wild-type, heterozygous EphA4+/-, and homozygous EphA4-/- mice were used in behavioral and in vivo electrophysiological investigations. We found that EphA4 knock-out (-/-) mice displayed impaired goal-directed reaching movements. In vivo intracellular recordings from forelimb motor neurons demonstrated increased corticoreticulospinal excitation, decreased direct reticulospinal excitation, and reduced direct propriospinal excitation in EphA4 knock-out mice. Cerebellar surface recordings showed a functional perturbation of the lateral reticular nucleus-cerebellum internal feedback pathway in EphA4 knock-out mice. Together, our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts the function of both feedforward and feedback motor pathways, resulting in deficits in skilled reaching.SIGNIFICANCE STATEMENT The central advances of this study are the demonstration that null mutation in the axon guidance molecule EphA4 gene impairs the ability of mice to perform skilled reaching, and identification of how these behavioral deficits correlates with discrete neurophysiological changes in central motor pathways involved in the control of reaching. Our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts both feedforward and feedback motor pathways, resulting in deficits in skilled reaching. This analysis of motor circuit function may help to understand the pathophysiological mechanisms underlying movement disorders in humans.

Human urine-derived stem cell-derived exosomal miR-21-5p promotes neurogenesis to attenuate Rett syndrome via the EPha4/TEK axis.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual-luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine-derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1)+ nerve cells as well as the transcription levels of ß-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX+ cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.

The developmental and genetic basis of 'clubfoot' in the peroneal muscular atrophy mutant mouse.

Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.

Inter-rhombomeric interactions reveal roles for fibroblast growth factors signaling in segmental regulation of EphA4 expression.

BACKGROUND: The basic ground plan of vertebrate hindbrain is established through a process of segmentation, which generates eight transient lineage-restricted cellular compartments called rhombomeres (r). The segments adopt distinct individual identities in response to axial patterning signals. It is unclear whether signaling between rhombomeres plays a conserved role in regulating segmental patterning during hindbrain development. RESULTS: Using tissue manipulations of rhombomeres in chicken embryos, we have uncovered roles for r2 and r4 in regulating the expression of EphA4 in r3 and r5. Perturbations of signaling pathways reveal that these regulatory inputs from r2 and r4 into EphA4 expression are mediated independent of inputs from Krox20 through cues involving fibroblast growth factor (FGF) signaling. These interactions are stage dependent and are set up in embryos with <10 somites. CONCLUSIONS: We show that r2 and r4 function as temporally dynamic signaling centers in the early patterning of adjacent hindbrain segments and this activity is dependent upon the FGF pathway. These results reveal that inter-rhombomeric signaling is a conserved feature of the regulatory networks that control the specification of individual rhombomere identities in vertebrate hindbrain segmentation. However, the timing of when restricted domains of FGF signaling are coupled to formation of r4 may vary between the species.

miR-455-3p alleviates propofol-induced neurotoxicity by reducing EphA4 expression in developing neurons.

PURPOSE: Propofol, an aesthetic agent in paediatric patients, results in neurotoxicity in the developing neurons. To reduce side effects of propofol, the protective role of miR-455-3p (microRNA-455-3p) in developing rat brain was investigated. MATERIALS AND METHODS: Primary hippocampal neurons were isolated from postnatal day 1 or 2 SD (Sprague-Dawley) rats. The neurons were exposed to various concentrations of propofol (0, 10, 30, or 50 µM) for 6 h. Propofol-induced cell viability was assessed by MTT assay, expression levels of miR-455-3p and EphA4 (erythropoietin-producing hepatocellular A4) in propofol-induced neurons were determined using qRT-PCR and western blot, respectively. Binding ability between miR-455-3p and EphA4 was predicted, and then validated by luciferase reporter assay. Neurons expressing miR-455-3p mimics, were treated with 50 µM propofol for 6 h, and apoptosis status was evaluated by flow cytometry. RESULTS: Exposure to propofol significantly decreased cell viability of developing neurons isolated from neonatal rats. Propofol decreased miR-455-3p expression, while increased EphA4 level in the neurons. miR-455-3p mimics increased propofol-induced reduce in cell viability, and attenuated propofol-induced cell apoptosis of neurons. MiR-455-3p could target EphA4, and decreased expression of EphA4 in neurons exposure to propofol. EphA4 knockdown counteracted with the promotive effects of propofol on cell viability and apoptosis of neurons. CONCLUSION: Propofol treatment induces neurotoxicity and suppresses miR-455-3p levels in the developing hippocampal neurons. However, miR-455-3p could alleviate such neurotoxicity by reducing EphA4 expression, provided new insights into miR-455-3p as novel therapeutic target to prevent propofol-induced damages from bench to clinic.

EphA4 Regulates Hippocampal Neural Precursor Proliferation in the Adult Mouse Brain by d-Serine Modulation of N-Methyl-d-Aspartate Receptor Signaling.

The hippocampal dentate gyrus (DG) is a major region of the adult rodent brain in which neurogenesis occurs throughout life. The EphA4 receptor, which regulates neurogenesis and boundary formation in the developing brain, is also expressed in the adult DG, but whether it regulates adult hippocampal neurogenesis is not known. Here, we show that, in the adult mouse brain, EphA4 inhibits hippocampal precursor cell proliferation but does not affect precursor differentiation or survival. Genetic deletion or pharmacological inhibition of EphA4 significantly increased hippocampal precursor proliferation in vivo and in vitro, by blocking EphA4 forward signaling. EphA4 was expressed by mature hippocampal DG neurons but not neural precursor cells, and an EphA4 antagonist, EphA4-Fc, did not activate clonal cultures of precursors until they were co-cultured with non-precursor cells, indicating an indirect effect of EphA4 on the regulation of precursor activity. Supplementation with d-serine blocked the increased precursor proliferation induced by EphA4 inhibition, whereas blocking the interaction between d-serine and N-methyl-d-aspartate receptors (NMDARs) promoted precursor activity, even at the clonal level. Collectively, these findings demonstrate that EphA4 indirectly regulates adult hippocampal precursor proliferation and thus plays a role in neurogenesis via d-serine-regulated NMDAR signaling.

The Transcription Factor LHX1 Regulates the Survival and Directed Migration of POA-derived Cortical Interneurons.

The delicate balance of excitation and inhibition is crucial for proper function of the cerebral cortex, relying on the accurate number and subtype composition of inhibitory gamma-aminobutyric (GABA)-expressing interneurons. Various intrinsic and extrinsic factors precisely orchestrate their multifaceted development including the long-range migration from the basal telencephalon to cortical targets as well as interneuron survival throughout the developmental period. Particularly expressed guidance receptors were described to channel the migration of cortical interneurons deriving from the medial ganglionic eminence (MGE) and the preoptic area (POA) along distinct routes. Hence, unveiling the regulatory genetic networks controlling subtype-specific gene expression profiles is key to understand interneuron-specific developmental programs and to reveal causes for associated disorders. In contrast to MGE-derived interneurons, little is known about the transcriptional networks in interneurons born in the POA. Here, we provide first evidence for the LIM-homeobox transcription factor LHX1 as a crucial key player in the post-mitotic development of POA-derived cortical interneurons. By transcriptional regulation of related genes, LHX1 modulates their survival as well as the subtype-specific expression of guidance receptors of the Eph/ephrin family, thereby affecting directional migration and layer distribution in the adult cortex.

Peripheral loss of EphA4 ameliorates TBI-induced neuroinflammation and tissue damage.

BACKGROUND: The continuum of pro- and anti-inflammatory response elicited by traumatic brain injury (TBI) is suggested to play a key role in the outcome of TBI; however, the underlying mechanisms remain ill -defined. METHODS: Here, we demonstrate that using bone marrow chimeric mice and systemic inhibition of EphA4 receptor shifts the pro-inflammatory milieu to pro-resolving following acute TBI. RESULTS: EphA4 expression is increased in the injured cortex as early as 2 h post-TBI and on CX3CR1gfp-positive cells in the peri-lesion. Systemic inhibition or genetic deletion of EphA4 significantly reduced cortical lesion volume and shifted the inflammatory profile of peripheral-derived immune cells to pro-resolving in the damaged cortex. These findings were consistent with in vitro studies showing EphA4 inhibition or deletion altered the inflammatory state of LPS-stimulated monocyte/macrophages towards anti-inflammatory. Phosphoarray analysis revealed that EphA4 may regulate pro-inflammatory gene expression by suppressing the mTOR, Akt, and NF-κB pathways. Our human metadata analysis further demonstrates increased EPHA4 and pro-inflammatory gene expression, which correlates with reduced AKT concurrent with increased brain injury severity in patients. CONCLUSIONS: Overall, these findings implicate EphA4 as a novel mediator of cortical tissue damage and neuroinflammation following TBI.

Amyloid-ß oligomers synaptotoxicity: The emerging role of EphA4/c-Abl signaling in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by progressive memory loss and dementia. The strong correlation between cognitive decline and the loss of synapses supports the idea that synaptic damage is a relevant pathogenic mechanism underlying AD progression. It has been shown that amyloid beta oligomers (AßOs) induce synaptotoxicity ultimately leading to the reduction of dendritic spine density, which underlies cognitive damage. However, the signaling pathways connecting AßOs to synaptic dysfunction have not been completely elucidated. In this review, we have gathered evidence on AßOs receptors and the signaling pathways involved in synaptic damage. We make special emphasis on a new AßOs induced axis that involves the tyrosine kinase ephrin receptor A4 (EphA4) and c-Abl tyrosine kinase activation. EphA4 is a key player in homeostatic plasticity, mediating dendritic spine remodeling and retraction. AßOs aberrantly activate EphA4 leading to dendritic spine elimination. c-Abl is activated in AßOs exposed neurons and in AD patient's brain, and the inhibition of activated c-Abl ameliorates cognitive deficits in AD mouse model. The EphA4 receptor activates c-Abl intracellular signaling. Therefore EphA4 is an emerging AßOs receptor and the activation of the EphA4/c-Abl axis would explain the synaptic spine alterations found in AD.

Regulation of EBV LMP1-triggered EphA4 downregulation in EBV-associated B lymphoma and its impact on patients' survival.

Epstein-Barr virus (EBV), an oncogenic human virus, is associated with several lymphoproliferative disorders, including Burkitt lymphoma, Hodgkin disease, diffuse large B-cell lymphoma (DLBCL), and posttransplant lymphoproliferative disorder (PTLD). In vitro, EBV transforms primary B cells into lymphoblastoid cell lines (LCLs). Recently, several studies have shown that receptor tyrosine kinases (RTKs) play important roles in EBV-associated neoplasia. However, details of the involvement of RTKs in EBV-regulated B-cell neoplasia and malignancies remain largely unclear. Here, we found that erythropoietin-producing hepatocellular receptor A4 (EphA4), which belongs to the largest RTK Eph family, was downregulated in primary B cells post-EBV infection at the transcriptional and translational levels. Overexpression and knockdown experiments confirmed that EBV-encoded latent membrane protein 1 (LMP1) was responsible for this EphA4 suppression. Mechanistically, LMP1 triggered the extracellular signal-regulated kinase (ERK) pathway and promoted Sp1 to suppress EphA4 promoter activity. Functionally, overexpression of EphA4 prevented LCLs from proliferation. Pathologically, the expression of EphA4 was detected in EBV(-) tonsils but not in EBV(+) PTLD. In addition, an inverse correlation of EphA4 expression and EBV presence was verified by immunochemical staining of EBV(+) and EBV(-) DLBCL, suggesting EBV infection was associated with reduced EphA4 expression. Analysis of a public data set showed that lower EphA4 expression was correlated with a poor survival rate of DLBCL patients. Our findings provide a novel mechanism by which EphA4 can be regulated by an oncogenic LMP1 protein and explore its possible function in B cells. The results provide new insights into the role of EphA4 in EBV(+) PTLD and DLBCL.

A novel miR17/protein tyrosine phosphatase-oc/EphA4 regulatory axis of osteoclast activity.

Information about the molecular mechanisms leading to the activation of the osteoclast is relatively limited. While there is compelling evidence that the signaling mechanisms of Src and integrin ß3 are essential for osteoclast activation, the regulation of these two signaling mechanisms is not fully understood. In this review, evidence supporting a novel regulatory axis of osteoclast activation that plays an upstream regulatory role in both the Src and integrin ß3 signaling during osteoclast activation is discussed. This regulatory axis contains three unique components: a structurally unique transmembrane protein-tyrosine phosphatase, PTP-oc, EphA4, and miR17. In the first component, PTP-oc activates the Src signaling through dephosphorylation of the inhibitory tyr-527 of Src. This in turn activates the integrin ß3 signaling, enhances the JNK2/NFκB signaling, promotes the ITAM/Syk signaling, and suppresses the ITIM/Shp1 signaling; the consequence of which is activation of the osteoclast. In the second component, EphA4 inhibits osteoclast activity by suppressing the integrin ß3 signaling. PTP-oc relieves the suppressive actions of EphA4 by directly dephosphorylating EphA4. In the third component, PTP-oc expression is negatively regulated by miR17. Accordingly, suppression of miR17 during osteoclast activation upregulates the PTP-oc signaling and suppresses the EphA4 signaling, resulting in the activation of the osteoclast. This regulatory axis is unique, in that each of the three components acts to exert suppressive action on their respective immediate downstream inhibitory step. Because the final downstream event is the EphA4-mediated inhibition of osteoclast activation, the overall effect of this mechanism is the stimulation of osteoclast activity.

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.

The Role of EphA4 Signaling in Radiation-Induced EMT-Like Phenotype in Colorectal Cancer Cells.

Radiotherapy is widely used for advanced rectal tumors. However, refractory metastasis has become the major cause of therapy failure in rectal cancer patients. Understanding the molecular mechanism that controls the aggressive cellular response to this treatment is essential for developing new therapeutic applications and improving radiotherapy response in colorectal cancer patients. Using the progeny of cells that were submitted to irradiation, we have demonstrated that the PI3K/AKT, Wnt/ß-catenin signaling pathways as well as ERK1/2 downstream of EPHA4 receptor activation, play an important role in the regulation of events related with the EMT development, which may be associated with the therapeutic failure in rectal cancer after radiotherapy. Here, we further discuss about EphA4 receptor as a potential therapeutic target for the treatment of this cancer type. J. Cell. Biochem. 118: 442-445, 2017. © 2016 Wiley Periodicals, Inc.

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.