Regulatory roles of ephrinA5 and its novel signaling pathway in mouse primary granulosa cell apoptosis and proliferation

Recent findings suggest that ephrinA5 (Efna5) has a novel role in female mouse fertility, in addition to its well-defined role as a neurogenesis factor. Nevertheless, its physiological roles in ovarian granulosa cells (GC) have not been determined. In this study, mouse GC were cultured and transfected with ephrin A5 siRNA and negative control to determine the effects of Efna5 on GC apoptosis, proliferation, cell cycle progression, and related signaling pathways. To understand the mode signaling, the mRNA expression levels of Efna5 receptors (Eph receptor A5, Eph receptor A3, Eph receptor A8, and Eph receptor B2) were examined. Both mRNA and protein expressions of apoptosis-related factors (Bax, Bcl-2, Caspase 8, Caspase 3, and Tnfα) and a proliferation marker, Pcna, were investigated. Additionally, the role of Efna5 on paracrine oocyte-secreted factors and steroidogenesis hormones were also explored. Efna5 silencing suppressed GC apoptosis by downregulating Bax and upregulating Bcl-2 in a Caspase 8-dependent manner. Efna5 knockdown promoted GC proliferation via p-Akt and p-ERK pathway activation. The inhibition of Efna5 enhanced BMH15 and estradiol expression, but suppressed GDF9, while progesterone level remained unaltered. These results demonstrated that Efna5 is a pro-apoptotic agent in GC and plays important role in folliculogenesis by mediating apoptosis, proliferation, and steroidogenesis in female mouse. Therefore Efna5 might be potential therapeutic target for female fertility disorders.     

Characterization of ephrin-A1 and ephrin-A4 as ligands for the EphA8 receptor protein tyrosine kinase.

The Eph receptors are the largest known family of receptor protein tyrosine kinases, which play important roles with their ligands called ephrin in the neural development, angiogenesis, and vascular network assembly. It was previously shown that ephrin-A2, -A3 and -A5 bind to, and activate the EphA8 receptor tyrosine kinase, respectively. In this study, we have examined if there are other additional ephrin ligands interacting with the EphA8 receptor tyrosine kinase expressed in NIH3T3 fibroblasts. For this purpose, we have constructed chimeric ephrin-A1, -A4, -B1, -B2 or -B3 ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. Both ephrin-A1 and ephrin-A4 chimeric ligands efficiently bound to the EphA8 receptor expressed in NIH3T3 fibroblasts, whereas the transmembrane ligands including ephrin-B1, -B2 and -B3 did not. Additionally we have demonstrated that both the EphA8-TrkB chimeric receptor and the EphA8 receptor expressed in NIH3T3 fibroblasts are efficiently tyrosine-phosphorylated upon stimulating with epthin-A1 or -A4 but none of transmembrane ephrin-B proteins. These results strongly indicate that the EphA8 receptor functions exclusively as an glycosyl phosphatidylinositol (GPI)-linked ephrin ligand-dependent receptor protein tyrosine kinase.     

Expression and function of the Eph A receptors and their ligands ephrins A in the rat thymus

Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.     

Expression Profile and Role of EphrinA1 Ligand After Spinal Cord Injury

Spinal cord injury (SCI) triggers the re-expression of inhibitory molecules present in early stages of development, contributing to prevention of axonal regeneration. Upregulation of EphA receptor tyrosine kinases after injury suggest their involvement in the nervous system’s response to damage. However, the expression profile of their ephrinA ligands after SCI is unclear. In this study, we determined the expression of ephrinA ligands after contusive SCI. Adult Sprague-Dawley female rats were injured using the MASCIS impactor device at the T10 vertebrae, and levels of ephrinA mRNA and protein determined at different time points. Identification of the cell phenotype expressing the ephrin ligand and colocalization with Eph receptors was performed with immunohistochemistry and confocal microscopy. Behavioral studies were made, after blocking ephrinA1 expression with antisense (AS) oligonucleotides, to assess hindlimb locomotor activity. Real-time PCR demonstrated basal mRNA levels of ephrin (A1, A2, A3, and A5) in the adult spinal cord. Interestingly, ephrinA1 was the only ligand whose mRNA levels were significantly altered after SCI. Although ephrinA1 mRNA levels increased after 2 weeks and remain elevated, we did not observe this pattern at the protein level as revealed by western blot analysis. Immunohistochemical studies showed ephrinA1 expression in reactive astrocytes, axons, and neurons and also their colocalization with EphA4 and A7 receptors. Behavioral studies revealed worsening of locomotor activity when ephrinA1 expression was reduced. This study suggests that ephrinA1 ligands play a role in the pathophysiology of SCI.     

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

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

The divergent C. elegans ephrin EFN-4 functions inembryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor

The C. elegans genome encodes a single Eph receptor tyrosine kinase, VAB-1, which functions in neurons to control epidermal morphogenesis. Four members of the ephrin family of ligands for Eph receptors have been identified in C. elegans. Three ephrins (EFN-1/VAB-2, EFN-2 and EFN-3) have been previously shown to function in VAB-1 signaling. We show that mutations in the gene mab-26 affect the fourth C. elegans ephrin, EFN-4. We show that efn-4 also functions in embryonic morphogenesis, and that it is expressed in the developing nervous system. Interestingly, efn-4 mutations display synergistic interactions with mutations in the VAB-1 receptor and in the EFN-1 ephrin, indicating that EFN-4 may function independently of the VAB-1 Eph receptor in morphogenesis. Mutations in the LAR-like receptor tyrosine phosphatase PTP-3 and in the Semaphorin-2A homolog MAB-20 disrupt embryonic neural morphogenesis. efn-4 mutations synergize with ptp-3 mutations, but not with mab-20 mutations, suggesting that EFN-4 and Semaphorin signaling could function in a common pathway or in opposing pathways in C. elegans embryogenesis.     

Multiple ephrins control cell organization in C. elegans using kinase-dependent and -independent functions of the VAB-1 Eph receptor

Eph receptor (EphR) tyrosine kinases and their ephrin ligands mediate direct cell-to-cell signaling. The C. elegans genome encodes four potential GPI-modified ephrins (EFN-1 to -4) and one EphR (VAB-1). Single and multiple ephrin mutants reveal functions for EFN-1, EFN-2, and EFN-3 in epidermal cell organization that, in aggregate, mirror those of VAB-1. Ephrin mutants have defects in head morphology and enclosure of the embryo by the epidermis and identify ephrin-EphR signaling functions involved in aligning and fusing tail and head epidermal cells, respectively. Biochemical analyses indicate that EFN-1, EFN-2, and EFN-3 jointly activate the VAB-1 tyrosine kinase in vivo. Mutant phenotypes and expression pattern analysis suggest that multiple ephrins are involved in distinct aspects of kinase-dependent and kinase-independent VAB-1 signaling required for proper cell organization during development in C. elegans.     

Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.     

Tumor necrosis factor-alpha induction of endothelial ephrin A1 expression is mediated by a p38 MAPK- and SAPK/JNK-dependent but nuclear factor-kappa B-independent mechanism

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that induces a broad spectrum of responses including angiogenesis. Angiogenesis promoted by TNF-alpha is mediated, at least in part, by ephrin A1, a member of the ligand family for Eph receptor tyrosine kinases. Although TNF-alpha induces ephrin A1 expression in endothelial cells, the signaling pathways mediating ephrin A1 induction remain unknown. In this study, we investigated the signaling mechanisms of TNF-alpha-dependent induction of ephrin A1 in endothelial cells. Both TNFR1 and TNFR2 appear to be involved in regulating ephrin A1 expression in endothelial cells, because neutralizing antibodies to either TNFR1 or TNFR2 inhibited TNF-alpha-induced ephrin A1 expression. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by a trans-dominant inhibitory isoform of mutant IkappaBalpha did not affect ephrin A1 induction, suggesting that NF-kappaB proteins are not major regulators of ephrin A1 expression. In contrast, ephrin A1 induction was blocked by inhibition of p38 mitogen-activated protein kinase (MAPK) or SAPK/JNK, but not p42/44 MAPK, using either selective chemical inhibitors or dominant-negative forms of p38 MAPK or TNF receptor-associated factor 2. These findings indicate that TNF-alpha-induced ephrin A1 expression is mediated through JNK and p38 MAPK signaling pathways. Taken together, the results of our study demonstrated that induction of ephrin A1 in endothelial cells by TNF-alpha is mediated through both p38 MAPK and SAPK/JNK, but not p42/44 MAPK or NF-kappaB, pathways.     

Ligand recognition by A‐class Eph receptors: crystal structures of the EphA2 ligand‐binding domain and the EphA2/ephrin‐A1 complex

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex between EphA2 and ephrin‐A1 and of unbound EphA2. Although these structures are similar overall to their B‐class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A‐class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a ‘lock‐and‐key’‐type binding mechanism, in contrast to the ‘induced fit’ mechanism defining the B‐class molecules. This model is supported by structure‐based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell‐based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2‐specific homotypic cell adhesion interactions.     

Eph/ephrin signaling in epithelial development and homeostasis

Eph receptors and ephrin ligands are widely expressed during embryonic development with well-defined functions in directing neuronal and vascular network formation. Over the last decade, evidence has mounted that Ephs and ephrins are also actively involved in prenatal and postnatal development of epithelial tissues. Their functions beyond developmental settings are starting to be recognized as well. The diverse functions of Eph/ephrin are largely related to the complementary expression pattern of the Eph receptors and corresponding ephrin ligands that are expressed in adjacent compartments, although overlapping expression pattern also exists in epithelial tissue. The interconnection between Ephs or ephrins and classical cell junctional molecules suggests they may function coordinately in maintaining epithelial structural integrity and homeostasis. This review will highlight cellular and molecular evidence in current literature that support a role of Eph/ephrin systems in regulating epithelial cell development and physiology.     

The proangiogenic factor ephrin-A1 is up-regulated in radioresistant murine tumor by irradiation

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3'], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.     

Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. Genetic evidence suggests that ephrins may transduce signals and become tyrosine phosphorylated during embryogenesis. However, the induction and functional significance of ephrin phosphorylation is not yet clear. Here, we report that when we used ectopically expressed proteins, we found that an activated fibroblast growth factor (FGF) receptor associated with and induced the phosphorylation of ephrin B1 on tyrosine. Moreover, this phosphorylation reduced the ability of overexpressed ephrin B1 to reduce cell adhesion. In addition, we identified a region in the cytoplasmic tail of ephrin B1 that is critical for interaction with the FGF receptor; we also report FGF-induced phosphorylation of ephrins in a neural tissue. This is the first demonstration of communication between the FGF receptor family and the Eph ligand family and implicates cross talk between these two cell surface molecules in regulating cell adhesion.     

Promiscuous and specific recognition among ephrins and Eph receptors

Eph–ephrin interactions control the signal transduction between cells and play an important role in carcinogenesis and other diseases. The interactions between Eph receptors and ephrins of the same subclass are promiscuous; there are cross-interactions between some subclasses, but not all. To understand how Eph–ephrin interactions can be both promiscuous and specific, we investigated sixteen energy landscapes of four Eph receptors (A2, A4, B2, and B4) interacting with four ephrin ligands (A1, A2, A5, and B2). We generated conformational ensembles and recognition energy landscapes starting from separated Eph and ephrin molecules and proceeding up to the formation of Eph–ephrin complexes. Analysis of the Eph–ephrin recognition trajectories and the co-evolution entropy of 400 ligand binding domains of Eph receptor and 241 ephrin ligands identified conserved residues during the recognition process. Our study correctly predicted the promiscuity and specificity of the interactions and provided insights into their recognition. The dynamic conformational changes during Eph–ephrin recognition can be described by progressive conformational selection and population shift events, with two dynamic salt bridges between EphB4 and ephrin-B2 contributing to the specific recognition. EphA3 cancer-related mutations lowered the binding energies. The specificity is not only controlled by the final stage of the interaction across the protein–protein interface, but also has large contributions from binding kinetics with the help of dynamic intermediates along the pathway from the separated Eph and ephrin to the Eph–ephrin complex.     

Ephrin A5 expression promotes invasion and transformation of murine fibroblasts

Emerging evidence suggests involvement of the ephrin/Eph receptor system in tumourigenesis. Research on this new role has centred on the contribution of Eph receptors. In contrast, we focused on the elucidation of the role of ephrins, specifically ephrin A5. Results indicated an increase in invasive potential of ephrin A5-expressing murine fibroblasts, which was abolished by addition of a Src family kinase inhibitor. Furthermore, anchorage-independent growth was increased in ephrin A5-expressing cells. Stimulation with EphA5-Fc receptor increased colony size, but not colony number in ephrin A5 transfectants. Moreover, we observed morphogenetic transformation of ephrin A5-expressing 3T3 cells into a branching network when plated onto Matrigel. This behaviour was specific to ephrin A5 transfectants, as 3T3 cells expressing ephrin B1 displayed a phenotype similar to control 3T3 cells. We conclude that ectopic expression of ephrin A5 in murine fibroblasts elevates oncogenic potential, including increased invasive behaviour, anchorage-independent growth, and morphological transformation.