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PubMed2000

Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors.

Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

Amino Acid Motifs↗

PubMed2006

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases.

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.

Amino Acid Substitution↗

PubMed2006

Comparative integromics on Eph family.

EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4 and EPHB6 are EPH family receptors for Ephrin family ligands. Ephrin/EPH signaling pathway networks with the WNT signaling pathway during embryogenesis, tissue regeneration, and carcinogenesis. TCF/LEF-binding sites within the promoter region of human EPH family members were searched for by using bioinformatics and human intelligence. Because five TCF/LEF-binding sites were identified within the 5'-promoter region of the EPHA7 gene, comparative genomics analyses on EPHA7 orthologs were further performed. EPHA7-MANEA-FHL5 locus at human chromosome 6q16.1 and EPHA10-MANEAL-FHL3 locus at human chromosome 1p34.3 were paralogous regions within the human genome. Human EPHA7 mRNA was expressed in embryonic stem (ES) cells, neural tissues, duodenal cancer and parathyroid tumors, while mouse Epha7 mRNA was expressed in fertilized egg, Rathke's pouche, visual cortex, pituitary gland, other neural tissues, pancreas, lung tumors and mammary tumors. The chimpanzee EPHA7 gene and cow Epha7 gene were identified within NW_107969.1 and AC155055.2 genome sequences, respectively. Five TCF/LEF-binding sites within human EPHA7 promoter were conserved in the chimpanzee EPHA7 promoter, and three TCF/LEF-binding sites in the cow Epha7 promoter, but none in the mouse Epha7 promoter. Primates and cow EPHA7 orthologs were identified as evolutionarily conserved targets of the WNT/beta-catenin signaling pathway. D6S1056 microsatellite marker within EPHA7 gene is deleted in prostate cancer. Deletion and/or promoter CpG hypermethylation could explain the EPHA7 down-regulation in human tumors. EPHA7 is a target of systems medicine, especially in the fields of regenerative medicine and oncology.

Animals↗

PubMed1998

PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.

3T3 Cells↗

PubMed2000

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Receptor regulation of gene expression of axon guidance molecules: implications for adaptation.

Axon guidance molecules, critical for neurodevelopment, are also implicated in morphological and other neurodaptative changes mediated by physiological or pharmacological events in adult brain. As an example, the psychostimulant cocaine markedly alters axon guidance molecules in adult brain of cocaine-treated rats. To decipher a potential link between drug-induced activation of G-protein-coupled receptors (GPCRs) and modulation of axon guidance molecules, we investigated whether GPCR activity in a SK-N-MC human neuroepithelioma cell line (which expresses low levels of D1 dopamine receptors) affects gene expression of axon guidance molecules (semaphorins, ephrins, netrins, and their receptors). Using real-time polymerase chain reaction, we identified 17 of 26 axon guidance molecules in these cells, with varying levels of expression. Forskolin, which raised intracellular cAMP levels 340%, increased EphA5, EphB2, and Neuropilin1 expression, paralleling reported changes in the rat hippocampus after cocaine treatment. The dopamine receptor agonist dihydrexidine, which raised cAMP levels 22%, promoted regulatory changes in EphrinA1, EphrinA5, EphB1, DCC, and Semaphorin3C, whereas (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF81297) altered EphA5, EphrinA1, EphrinA5, and neuropilin1. cAMP and other signal transduction pathways may regulate gene expression of axon guidance molecules, potentially linking monoamine receptor activation to signal transduction cascades, transcriptional regulation of axon guidance molecules, and alterations in neural networks.

Adaptation, Physiological↗

PubMed2006

The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization.

Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.

Actins↗

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↗

PubMed2003

B-type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways.

Forward and reverse signaling mediated by EphB tyrosine kinase receptors and their transmembrane ephrin-B ligands play important roles in axon pathfinding, yet little is known about the intracellular pathways involved. Here we have used growth cones from the ventral (EphB receptor-bearing) and dorsal (ephrin-B-bearing) embryonic Xenopus retina to investigate the signaling mechanisms in both forward and reverse directions. We report that unclustered, but not clustered, EphB2 ectodomains trigger fast (5-10 min) transient collapse responses in growth cones. This collapse response is mediated by low levels of intracellular cyclic GMP and requires proteasome function. In contrast, clustered, but not unclustered, ephrin-B1 ectodomains cause slow (30-60 min) growth cone collapse that depends on high cGMP levels and is insensitive to inhibition of the proteasomal pathway. Upon receptor-ligand binding, endocytosis occurs in the reverse direction (EphB2-Fc into dorsal retinal growth cones), but not the forward direction, and is also sensitive to proteasomal inhibition. Endocytosis is functionally important because blocking of EphB2 internalization inhibits growth cone collapse. Our data reveal that distinct signaling mechanisms exist for B-type Eph/ephrin-mediated growth cone guidance and suggest that endocytosis provides a fast mechanism for switching off signaling in the reverse direction.

Animals↗

PubMed2006

Structure and thermodynamic characterization of the EphB4/Ephrin-B2 antagonist peptide complex reveals the determinants for receptor specificity.

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 angstroms. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.

Binding Sites↗

PubMedDate not supplied

Growth factor receptors in hematopoietic stem cells: EPH family expression in CD34+ and CD133+ cell populations from mobilized peripheral blood.

Cell-surface antigen expression of hematopoietic stem cells has a crucial role in characterizing cell subpopulation with distinct functional properties. The Eph receptors are the largest receptor tyrosine kinase family being involved in processes like vascular remodelling during development and physiological and pathological angiogenesis. Some Eph/Ephrin members are expressed in hematopoietic cells. The ability to isolate purified cell populations co-expressing CD34 and CD133 antigens as most commonly used markers for identification of hematopoietic progenitors has provided the opportunity to identify their surface-receptor profile. As positively expressed CD34 and CD133 cells take place not only in hematopoietic but also in endothelial differentiation, we aimed to define the Eph/Ephrin characteristic of these cells and relate these findings to new therapy strategies. Positive selections of CD34 and CD133 cells from PBPC in lymphoma patients were performed using magnetic beads and AutoMACS (Miltenyi Biotec) device. The purity of isolated cells was tested by flow cytometry. Immunocytochemistry was used to assess the Eph/Ephrin expression profile of positively selected samples. Our study revealed that all samples (10 from CD34+ and 8 from CD133+ cells) expressed one or more of Eph/Ephrin antigens in different proportions. All CD34+ cell samples, and 6 of 8 in the CD133+ cell fraction were strongly immunoreactive for EphA2. EphB2 was strongly expressed in all CD133+ cases, but 50% of the CD34 positive group lacked or weakly expressed this receptor. EphB4 was negative in 9 of 10 CD34+ cases and in all CD133+ cells. Thus, we have shown the surface marker profile of positively selected CD34 and CD133 cells in leukapheresis samples from lymphoma patients with regard to Eph/Ephrin receptors and discussed their biological clinical potential.

AC133 Antigen↗

PubMed2002

Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB.

In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. We show here that beta-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3(-/-) mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. We conclude that in the intestinal epithelium beta-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system.

Actins↗

PubMed2006

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Cell Culture Techniques↗

PubMed2003

Analysis of EphB receptors and their ligands in the developing retinocollicular system of the wallaby reveals dynamic patterns of expression in the retina.

The expression of EphB1 and B2 receptors and ephrins-B1, -B2 and -B3 in the retina and superior colliculus of the wallaby (Macropus eugenii) was examined during the development of the retinocollicular projection, using reverse transcription-polymerase chain reaction and immunohistochemistry. There was an early transient differential expression of EphB2 that was higher in ventral retina and restricted to the outer neuroblast layer, whereas a high ventral to low dorsal gradient of ephrin-B2 expression occurred there throughout the study period. However, there was no dorsoventral gradient of receptors or ligands in retinal ganglion cells or a mediolateral gradient of ephrins in the colliculus. These findings suggest a limited role for these molecules in topographic mapping across the mediolateral colliculus in the wallaby. Early in retinal development there is a complementary pattern of expression of ephrin-B1 and -B2 in the outer neuroblast layer that overlaps with expression of EphB2. Ganglion and amacrine cells also express EphB2. As development proceeds subpopulations of putative horizontal and bipolar cells, also expressing EphB2, come to reside in the inner nuclear layer and ephrin-B1 is expressed throughout the outer nuclear layer. At the same time cells expressing ephrin-B2, and subpopulations of horizontal and bipolar cells come to reside in the inner nuclear layer and there is a corresponding decrease in ephrin-B2 expression in the outer nuclear layer. This pattern of coexpression of receptors and ligands suggests a role for them in cell migration and maintenance of laminar boundaries.

Aging↗

PubMed2000

Genomic structure and promoter analysis of the mouse EphA8 receptor tyrosine kinase gene.

The gene encoding the mouse EphA8 receptor tyrosine kinase has been isolated from a mouse genomic library, and its complete genomic structure has been determined. This gene spans approximately 28 kb and consists of 17 exons. This gene structure is similar to the structure of the chick EphB2 (Cek5) gene, except for one intron present between the first two exons encoding the EphA8 kinase domain. This difference may reflect an evolutionary divergence of the catalytic domain between EphA and EphB subgroup receptors. The site for transcription initiation has been mapped to the 19th nucleotide upstream from the translation start codon ATG. A feature of this gene is an unmethylated CpG island spanning exon 1 and the flanking sequence. The putative promoter of the EphA8 gene lacks a TATA box and contains multiple copies of the sequence GGGCGG, the core sequence of the putative Sp1-binding site. The 3.5-kb upstream genomic region containing part of the first exon showed strong promoter activity in NG108-15 neuroblastoma cells but much less in 293T cells, suggesting that this fragment is sufficient for neural cell-directed promoter activity. By deleting the genomic region containing the five GC boxes, it was shown that the minimal promoter region is primarily comprised of five copies of the Sp1-binding site located upstream from the transcription initiation site. Finally, in situ RNA hybridization studies revealed a very specific pattern of EphA8 gene expression restricted to the rostral region of midbrain tectum during embryonic development. Isolation of a functional promoter for the EphA8 gene is a first step in understanding how expression of this gene is controlled at the molecular level.

Amino Acid Sequence↗

PubMed2006

Ephrin-B3 ligand promotes glioma invasion through activation of Rac1.

Eph receptor tyrosine kinases are involved in nervous system development. Eph ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in glioma cells increases cell invasion. Here, expression profiles of ephrin-B family members were determined in four glioma cell lines and in invading glioblastoma cells collected by laser capture microdissection. Ephrin-B3 mRNA was up-regulated in migrating cells of four of four glioma cell lines (1.3- to 1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251, SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA negated the increased invasion by addition of EphB2/Fc. In human glioma specimens, ephrin-B3 expression and phosphorylation correlated with increasing tumor grade. Immunohistochemistry revealed robust staining for phosphorylated ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that ephrin-B3 expression and signaling through Rac1 are critically important to glioma invasion.

Animals↗

PubMed2005

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro.

AIM: Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinA1, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin-B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD.

Crohn Disease↗

PubMed1999

Genomic structure of the EPHA1 receptor tyrosine kinase gene.

Some receptor tyrosine kinase genes are mutated in inherited and somatically acquired human cancers. To permit mutational analysis, the complete genomic structure of the human EPHA1 gene on chromosome 7q34 was determined and oligonucleotide pairs were designed to amplify coding regions. The gene contains 18 exons, two more than the related tyrosine kinase, EPHB2. Presumed sequencing errors in the published cDNA sequence of EPHA1 were identified in exons 10 and 11. Availability of this information will facilitate mutational analysis of EPHA1.

Base Sequence↗