Eph receptors are involved in the activity-dependent synaptic wiring in the mouse cerebellar cortex

Eph receptor tyrosine kinases are involved in many cellular processes. In the developing brain, they act as migratory and cell adhesive cues while in the adult brain they regulate dendritic spine plasticity. Here we show a new role for Eph receptor signalling in the cerebellar cortex. Cerebellar Purkinje cells are innervated by two different excitatory inputs. The climbing fibres contact the proximal dendritic domain of Purkinje cells, where synapse and spine density is low; the parallel fibres contact the distal dendritic domain, where synapse and spine density is high. Interestingly, Purkinje cells have the intrinsic ability to generate a high number of spines over their entire dendritic arborisations, which can be innervated by the parallel fibres. However, the climbing fibre input continuously exerts an activity-dependent repression on parallel fibre synapses, thus confining them to the distal Purkinje cell dendritic domain. Such repression persists after Eph receptor activation, but is overridden by Eph receptor inhibition with EphA4/Fc in neonatal cultured cerebellar slices as well as mature acute cerebellar slices, following in vivo infusion of the EphA4/Fc inhibitor and in EphB receptor-deficient mice. When electrical activity is blocked in vivo by tetrodotoxin leading to a high spine density in Purkinje cell proximal dendrites, stimulation of Eph receptor activation recapitulates the spine repressive effects of climbing fibres. These results suggest that Eph receptor signalling mediates the repression of spine proliferation induced by climbing fibre activity in Purkinje cell proximal dendrites. Such repression is necessary to maintain the correct architecture of the cerebellar cortex

An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly

Erythropoetin-producing hepatoma (Eph) receptors are cell-surface protein tyrosine kinases mediating cell-cell communication. Upon activation, they form signaling clusters. We report crystal structures of the full ectodomain of human EphA2 (eEphA2) both alone and in complex with the receptor-binding domain of the ligand ephrinA5 (ephrinA5 RBD). Unliganded eEphA2 forms linear arrays of staggered parallel receptors involving two patches of residues conserved across A-class Ephs. eEphA2-ephrinA5 RBD forms a more elaborate assembly, whose interfaces include the same conserved regions on eEphA2, but rearranged to accommodate ephrinA5 RBD. Cell-surface expression of mutant EphA2s showed that these interfaces are critical for localization at cell-cell contacts and activation-dependent degradation. Our results suggest a 'nucleation' mechanism whereby a limited number of ligand-receptor interactions 'seed' an arrangement of receptors which can propagate into extended signaling arrays

Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

10.1371/journal.pone.0074040PLoS ONE89-POLN

Differential Gene Expression in the EphA4 Knockout Spinal Cord and Analysis of the Inflammatory Response Following Spinal Cord Injury

Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips™. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury

EPHB2 germline variants in patients with colorectal cancer or hyperplastic polyposis

BACKGROUND: Ephrin receptor B2 (EPHB2) has recently been proposed as a novel tumor suppressor gene in colorectal cancer (CRC). Inactivation of the gene has been shown to correlate with progression of colorectal tumorigenesis, and somatic mutations have been reported in both colorectal and prostate tumors. METHODS: Here we have analyzed the EPHB2 gene for germline alterations in 101 individuals either with 1) CRC and a personal or family history of prostate cancer (PC), or 2) intestinal hyperplastic polyposis (HPP), a condition associated with malignant degeneration such as serrated adenoma and CRC. RESULTS: Four previously unknown missense alterations were observed, which may be associated with the disease phenotype. Two of the changes, I361V and R568W, were identified in Finnish CRC patients, but not in over 300 Finnish familial CRC or PC patients or more than 200 population-matched healthy controls. The third change, D861N, was observed in a UK HPP patient, but not in additional 40 UK HPP patients or in 200 UK healthy controls. The fourth change R80H, originally identified in a Finnish CRC patient, was also found in 1/106 familial CRC patients and in 9/281 healthy controls and is likely to be a neutral polymorphism. CONCLUSION: We detected novel germline EPHB2 alterations in patients with colorectal tumors. The results suggest a limited role for these EPHB2 variants in colon tumor predisposition. Further studies including functional analyses are needed to confirm this

Germline EPHB2 Receptor Variants in Familial Colorectal Cancer

Familial clustering of colorectal cancer occurs in 15–20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer

EphB6 Receptor Modulates Micro RNA Profile of Breast Carcinoma Cells

Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways

Diverse Roles of Eph/ephrin Signaling in the Mouse Lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells

The Synovial Sarcoma-Associated SYT-SSX2 Oncogene Antagonizes the Polycomb Complex Protein Bmi1

This study demonstrates deregulation of polycomb activity by the synovial sarcoma-associated SYT-SSX2 oncogene, also known as SS18-SSX2. Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation event that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. The role of the translocation products in this disease is poorly understood. We present evidence that the SYT-SSX2 fusion protein interacts with the polycomb repressive complex and modulates its gene silencing activity. SYT-SSX2 causes destabilization of the polycomb subunit Bmi1, resulting in impairment of polycomb-associated histone H2A ubiquitination and reactivation of polycomb target genes. Silencing by polycomb complexes plays a vital role in numerous physiological processes. In recent years, numerous reports have implicated gain of polycomb silencing function in several cancers. This study provides evidence that, in the appropriate context, expression of the SYT-SSX2 oncogene leads to loss of polycomb function. It challenges the notion that cancer is solely associated with an increase in polycomb function and suggests that any imbalance in polycomb activity could drive the cell toward oncogenesis. These findings provide a mechanism by which the SYT-SSX2 chimera may contribute to synovial sarcoma pathogenesis