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PubMed2006

EphB receptors coordinate migration and proliferation in the intestinal stem cell niche.

More than 10(10) cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche.

Adenoma↗
PubMed1998

Tyrosine-614, the major autophosphorylation site of the receptor tyrosine kinase HEK2, functions as multi-docking site for SH2-domain mediated interactions.

HEK2 belongs to the family of EPH-related receptor tyrosine kinases (RTK) which are involved in axonal pathfinding and the formation of the embryonic body plan. The knowledge about intracellular pathways of signal transduction mediated by EPH-related receptors is still limited. Many of the known key players of cellular signalling contain Src homology 2 (SH2) domains, which recognize phosphotyrosine motifs in RTKs. Thus, we examined the interactions of various SH2-containing molecules like PLC-gamma1, rasGAP, p85 subunit of PI3-kinase, Src, Fyn, Crk, Nck, Grb2 and Shc with HEK2 using in vitro binding assays, immunoprecipitations and yeast Two-Hybrid assays. We found that rasGAP, Crk and Fyn bind in a SH2-dependent manner to autophosphorylated HEK2. rasGAP, which contains two SH2- and one SH3-domain, was shown to associate with its N-terminal SH2-domain to HEK2. Furthermore, we demonstrated that a single amino acid substitution (Y614F) clearly reduces the phosphotyrosine content of HEK2 and abrogates its ability to bind rasGAP, Crk and Fyn indicating that this residue functions as major phosphorylation and multi-docking site. The conservation of this predicted binding site among various EPH-related RTKs provides evidence that Fyn, Crk and rasGAP are key players in signal transduction of at least a subset of these receptors.

Binding Sites↗
PubMed1996

Cell-cell adhesion mediated by binding of membrane-anchored ligand LERK-2 to the EPH-related receptor human embryonal kinase 2 promotes tyrosine kinase activity.

Human embryonal kinase 2 (HEK2) is a protein-tyrosine kinase that is a member of the EPH family of receptors. Transcripts for HEK2 have a wide tissue distribution. Recently, a still growing family of ligands, which we have named LERKs, for ligands of the eph-related kinases, has been isolated. In order to analyze functional effects between the LERKs and the HEK2 receptor, we expressed HEK2 cDNA in an interleukin-3-dependent progenitor cell line 32D that grows as single cells in culture. Within the group of LERKs, LERK-2 and -5 were shown to bind to HEK2. Membrane-bound and soluble forms of LERK-2 were demonstrated to signal through HEK2 as judged by receptor phosphorylation. Coincubation of HEK2 and LERK-2 expressing cells induced cell-cell adhesion and formation of cell aggregates. This interaction could be inhibited by preincubation of HEK2 expressing cells with soluble LERK-2. Coexpression of HEK2 and LERK-2 in 32D cells showed reduced kinase activity and autophosphorylation of HEK2 compared with the juxtacrine stimulation, which seems to be due to a reduced sensitivity of the receptor.

Animals↗
PubMed1997

Differentially regulated epithelial expression of an Eph family tyrosine kinase (fHek2) during tracheal surface airway and submucosal gland development.

A ferret model was used to evaluate the potential role of an Eph family tyrosine kinase (fHek2) in tracheal development of surface airway epithelium and submucosal glands. A partial 2.6-kb cDNA fragment of fHek2 was isolated from a ferret tracheal/lung cDNA library. Sequence analysis demonstrated that this gene is the ortholog to the previously cloned human Hek2 gene. In situ hybridization analysis of fHek2 mRNA expression on ferret tracheal developmental time points revealed an expression pattern within a subset of surface airway epithelial cells which remained relatively constant throughout tracheal development (from -2 d in utero to adult). In contrast, developing tracheal submucosal glands at 3-day postnatal time points demonstrated little fHek2 mRNA expression. However, expression of fHek2 significantly increased more than 4-fold over the course of gland development to adulthood. These findings, which demonstrate a uniquely regulated pattern of fHek2 mRNA expression between surface airway epithelium and submucosal glands, have implications on regulatory processes which control differentiation and/or maturation of secretory structures in the lung. Such findings may be useful in further delineating the mechanisms which control cellular differentiation in the lung and how these processes are abnormally regulated in hypersecretory diseases such as chronic bronchitis, asthma, and cystic fibrosis.

Amino Acid Sequence↗
PubMed1995

Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.

Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).

Amino Acid Sequence↗
PubMed1993

Five novel avian Eph-related tyrosine kinases are differentially expressed.

We have identified cDNA clones that encode five new avian receptor-like tyrosine kinases of the Eph subclass, by screening two chicken embryonic cDNA libraries with DNA probes. We have designated them Cek6 to Cek10. The identification of these kinases indicates that the Eph subclass comprises at least 10 members and, therefore, represents a very large family of receptor-like tyrosine kinases. Variants of Cek10 and of Cek5 (a previously identified Eph-related kinase) containing amino acid insertion sequences in the juxtamembrane domain were also isolated. The Cek5 variant is expressed in the brain, but not in other tissues of the 10-day chick embryo. Analysis of 10-day chick embryo mRNAs shows the newly identified tyrosine kinases to be all expressed in both the embryonic brain and body tissues. In adult tissues, they display distinct patterns of expression. Cek6, Cek7, Cek8, Cek9 and Cek10 are likely to play significant roles in embryonic signal transduction pathways, including those involved in neural development. Their distinct tissue distributions in the adult suggest that the different members of the Eph family may each serve specific functions.

Amino Acid Sequence↗
PubMed2004

Expression of ephrinB2 and its receptors on fibroproliferative membranes in ocular angiogenic diseases.

PURPOSE: To determine whether ephrinB2 plays a role in ocular angiogenesis, we investigated the expression of ephrinB2 and EphB receptors on retinal fibroproliferative membranes. DESIGN: Experimental study of the expression of ephrinB2 and EphB receptors within fibroproliferative membranes in patients with ocular angiogenic diseases collected during vitrectomy. METHODS: Fibroproliferative membranes were obtained at the time of vitrectomy from 20 patients with proliferative diabetic retinopathy (PDR) and from 40 patients who had stage 5 retinopathy of prematurity. Specimens were investigated with immunohistochemistry using polyclonal antibodies directed against ephrinB2 and the EphB2, EphB3, and EphB4 receptors. Immunoreactivity for von Willebrand factor (factor VIII) and alpha-smooth muscle actin (alpha-SMA) was also determined to confirm the identity of the target vascular endothelial cells. RESULTS: Positive staining for ephrinB2 was observed on fibroproliferative membranes that were obtained from patients with PDR (65.0%) and retinopathy of prematurity (25.0%). Specifically, ephrinB2 was found to be present on endothelial cells, as confirmed by its colocalization with factor VIII and alpha-SMA staining. EphB2 and EphB3 expression was observed on fibroproliferative membranes that were harvested from patients with PDR (EphB2, 90.0%; EphB3, 70.0%) and retinopathy of prematurity (EphB2, 35.0%; EphB3, 45.0%). However, EphB4 expression was not observed in any of the membranes derived from patients with PDR or retinopathy of prematurity. The rate of ephrinB2 expression in patients with PDR was significantly higher than that seen in patients with retinopathy of prematurity, which probably reflected differences in the vascular density of their fibroproliferative membranes. CONCLUSION: These data suggest that the ephrinB2-EphB2/B3 system may play an important role in ocular angiogenesis.

Actins↗
PubMed2003

Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus.

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2-mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.

Animals↗
PubMed2004

Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development.

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.

Anal Canal↗
PubMed2005

Expression of Eph receptors and their ligands, ephrins, during lipopolysaccharide fever in rats.

Erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinases and their ligands, ephrins, are involved in embryogenesis and oncogenesis by mediating cell adhesion and migration. Although ephrins can be induced by bacterial LPS in vitro, whether they are involved in inflammation in vivo is unknown. Using differential mRNA display, we found that a febrigenic dose of LPS (50 microg/kg iv) induces a strong transcriptional upregulation of ephrin-A1 in rat liver. We confirmed this finding by real-time RT-PCR. We then quantified the mRNA expression of different ephrins and Eph receptors at phases 1-3 of LPS fever in different organs. Febrile phases 2 (90 min post-LPS) and 3 (300 min) were characterized by robust upregulation (up to 16-fold) and downregulation (up to 21-fold) of several ephrins and Eph receptors. With the exception of EphA2, which showed upregulation in the brain at phase 2, expressional changes of Eph receptors and ephrins were limited to the LPS-processing organs: liver and lung. Characteristic, counter-directed changes in expressional regulation of Eph receptors and their corresponding ligands were found: upregulation of EphA2, downregulation of ephrin-A1 in the liver and lung at phase 2; downregulation of EphB3, upregulation of ephrin-B2 in the liver at phase 2; downregulation of EphA1 and EphA3, upregulation of ephrins-A1 and -A3 in liver at phase 3. In the liver, transcriptional changes of EphA2 and EphB3 at phase 2 were confirmed at protein level. These coordinated, phase-specific responses suggest that different sets of ephrins and Eph receptors may be involved in cellular events (such as disruption of tissue barriers and leukocyte transmigration) underlying different stages of systemic inflammatory response to LPS.

Animals↗
PubMed2006

A gene expression signature that distinguishes desmoid tumours from nodular fasciitis.

Nodular fasciitis (NF) is a rapidly growing cellular mass composed of fibroblasts/myofibroblasts, usually localized in subcutaneous tissues, that typically undergoes fibrosis and almost never recurs. Desmoid tumours (DTs) are rare forms of fibroblastic/myofibroblastic growth that arise in deep soft tissues, display a propensity for local infiltration and recurrence, but fail to metastasize. Given that both entities are primarily fibroblastic/myofibroblastic lesions with overlapping histological features, their gene expression profiles were compared to identify differentially expressed genes that may provide not only potential diagnostic markers, but also clues as to the pathogenesis of each disorder. Differentially expressed transcripts (89 clones displaying increased expression in DTs and 246 clones displaying increased expression in NF) included genes encoding several receptor and non-receptor tyrosine kinases (EPHB3, PTPRF, GNAZ, SYK, LYN, EPHA4, BIRC3), transcription factors (TWIST1, PITX2, EYA2, OAS1, MITF, TCF20), and members of the Wnt signalling pathway (AXIN2, WISP1, SFRP). Remarkably, almost one-quarter of the differentially expressed genes encode proteins associated with inflammation and tissue remodelling, including members of the interferon (IFN), tumour necrosis factor (TNF), and transforming growth factor beta (TGF-beta) signalling pathways as well as metalloproteinases (MMP1, 9, 13, 23), urokinase plasminogen activator (PLAU), and cathepsins. The observations provide the first comparative molecular characterization of desmoid tumours and nodular fasciitis and suggest that selected tyrosine kinases, transcription factors, and members of the Wnt, TGF-beta, IFN, and TNF signalling pathways may be implicated in influencing and distinguishing their fate.

Adolescent↗
PubMed2005

Retinoic acid regulates the expression of dorsoventral topographic guidance molecules in the chick retina.

Asymmetric expression of several genes in the early eye anlagen is required for the dorsoventral (DV) and anteroposterior (AP) patterning of the retina. Some of these early patterning genes play a role in determining the graded expression of molecules that are needed to form the retinotectal map. The polarized expression of retinoic acid synthesizing and degrading enzymes along the DV axis in the retina leads to several zones of varied retinoic acid (RA) activity. This is suggestive of RA playing a role in DV patterning of the retina. A dominant-negative form of the retinoic acid receptor alpha (DNhRARalpha) was expressed in the chick retina to block RA activity. RA signaling was found to play a role in regulating the expression of EphB2, EphB3 and ephrin B2, three molecules whose graded expression in the retina along the DV axis is important for establishing the correct retinotectal map. Blocking RA signaling by misexpression of a RA degrading enzyme, Cyp26A1 recapitulated some but not all the effects of DNhRARalpha. It also was found that Vax, a ventrally expressed transcription factor that regulates the expression of the EphB and ephrin B molecules, functions upstream of, or in parallel to, RA. Expression of DNhRARalpha led to increased levels of RA-synthesizing enzymes and loss of RA-degrading enzymes. Activation of such compensatory mechanisms when RA activity is blocked suggests that RA homeostasis is very strictly regulated in the retina.

Animals↗
PubMed2006

EphB3: an endogenous mediator of adult axonal plasticity and regrowth after CNS injury.

Endogenous mechanisms underlying the remodeling of neuronal circuitry after mammalian CNS injury or disease remain primarily unknown. Here, we investigated axonal plasticity after optic nerve injury and found that macrophages recruited into the injury site and adult retinal ganglion cell (RGC) axons, which undergo injury-induced sprouting and terminal remodeling, were linked by their respective expression of a ligand and receptor pair active in axon guidance. Recruited macrophages specifically upregulated mRNA encoding the guidance molecule EphB3 and expressed EphB proteins capable of binding Ephrin B molecules in vivo and in vitro. Injured adult RGC axons in turn expressed EphrinB3, a known receptor for EphB3, and RGC axons bound recombinant EphB3 protein injected into the optic nerve. In vitro, EphB3 supported adult RGC axon outgrowth, and axons turned toward a source of this guidance molecule. In vivo, both reduction of EphB3 function in adult heterozygous animals and loss of function in homozygous animals greatly decreased RGC axon re-extension or sprouting after optic nerve injury. Comparisons of axon re-extension in EphB3 null and wild-type littermates showed that this loss of axonal plasticity was not attributable to a difference in intrinsic axon growth potential. Rather, the results indicated an essential role for local optic nerve-derived EphB3 in regulating adult RGC axon plasticity after optic nerve injury. Of note, the loss of EphB3 did not affect the ability of injured RGC axons to elaborate complex terminal branching, suggesting that additional EphB3-independent mechanisms governed adult axon branching triggered by CNS damage.

Animals↗
PubMed2000

The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis.

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. Here we investigated the role of EphB receptors and their ligands during embryonic blood vessel development in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, we detected expression of EphB4 receptors in the posterior cardinal veins and their derivatives, the intersomitic veins. Vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could however not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Furthermore, we found that ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. We performed RNA injection experiments to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos resulted in intersomitic veins growing abnormally into the adjacent somitic tissue. Our findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis possibly by mediating repulsive guidance cues to migrating endothelial cells.

Animals↗
PubMed2000

Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk.

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

Animals↗
PubMed2005

Receptor related to tyrosine kinase RYK regulates cell migration during cortical development.

Mammalian RYK is a receptor related to tyrosine kinase without detectable catalytic activity. We have previously reported that rat RYK is dominantly expressed in neural progenitor cells and mature neurons in the developing central nervous system. Mouse RYK has been found to bind to EphB2/B3 receptors, which have diverse functions during development. In this study, we demonstrated that RYK, EphB2, EphB3, ephrinB1, and ephrinB2 are expressed in embryonic brain. In vitro analysis using COS-7 cells revealed binding between rat RYK and EphB3, and that the RYK deletion mutant without extracellular leucine-rich motifs lacked this binding ability. To investigate the function of RYK in vivo, embryonic cortical slice cultures were analyzed after electroporation of expression plasmids for RYK or its deletion mutants. The results showed that overexpression of RYK suppressed cell migration from the ventricular zone to the pial surface, however, overexpression of the RYK deletion mutant without leucine-rich motifs had no effect on cell migration. These results suggest that RYK regulates cell migration during mammalian cortical development through the binding to Eph receptors.

Animals↗