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PubMed2003

The msr/apj gene encoding the apelin receptor is an early and specific marker of the venous phenotype in the retinal vasculature.

Expression of the new G protein-coupled msr/apj receptor in the mouse embryo is restricted to the endothelial layer of the primary blood vessels and the newly forming heart (Mech. Dev. 84 (1999) 199). During development of the retinal vasculature, the msr/apj gene is not expressed throughout the vascular network, indicating a possible relationship between the localization of expression and the acquisition of arterial or venous identity (Mech. Dev. 110 (2002) 183). Here we first established that retinal expression of ephrin-B2 and its putative receptor EphB4 correlates with arterial and venous phenotype, respectively. Then we analyzed the expression pattern of msr/apj in the retinal vessels at various stages of postnatal development by in situ hybridization. In contrast to the expression of ephrin-B2 or EphB4, msr/apj transcripts can be detected as early as postnatal day P3. From P3 to P12, msr/apj expression in the vascular network is restricted to the venules and the associated capillaries. The msr/apj gene is thus an early and specific marker of the venous phenotype in the retinal vasculature.

Animals↗

PubMed2006

The prognostic impact of EphB2/B4 expression on patients with advanced ovarian carcinoma.

OBJECTIVES: To analyze expressions of the EphB2 and EphB4 receptors in ovarian carcinomas and explore their clinicopathological correlations and prognostic value. METHODS: 115 patients with advanced ovarian carcinoma FIGO IIB to IV were involved. RT-PCR and immunohistochemistry were used to examine the expressions of EphB2/B4 receptor mRNA and protein. Correlations between protein expression and clinicopathological factors were also analyzed. RESULTS: Ovarian carcinoma patients with age elder than 60 years had higher EphB2 expression than younger patients. Expression of EphB2 and EphB4 protein did not significantly correlate with any other clinical variables, including FIGO stage, residual tumor, histological type and differentiation grade. No significant correlation between mRNA and protein expression level for both of these receptors was seen. It was found that patients with strong immunostaining for EphB2 (P = 0.03) or EphB4 (P = 0.003) receptors had poorer survival, and patients with strong immunostaining for EphB4 receptor showed poorer response to chemotherapy (P = 0.036). CONCLUSIONS: These studies suggest that EphB2 and B4 receptors are of prognostic value and EphB4 receptor may be an independent predictor of chemotherapy response in ovarian cancer patients.

Adult↗

PubMed2006

Overexpression of ephrinB2 and EphB4 in tumor advancement of uterine endometrial cancers.

BACKGROUND: The ligand ephrinB2 and the corresponding receptor EphB4 contribute to tumor growth in various human tumors. This prompted us to study the expression and localization of ephrinB2 and EphB4 in uterine endometrial cancers to analyze the ephrinB2/EphB4 functions against clinical backgrounds. MATERIALS AND METHODS: We carried out immunohistochemistry and real-time RT-PCR to determine the histoscores and messenger RNA (mRNA) levels of ephrinB2 and EphB4, respectively, in 68 uterine endometrial cancers and 16 normal endometrium tissue samples. Patient prognoses were analyzed with a 60-month survival rate. RESULTS: The localization of ephrinB2 and EphB4 was dominantly in the cancer cells of uterine endometrial cancer of all cases given. EphrinB2 and EphB4 histoscores were highly correlated with ephrinB2 and EphB4 mRNA levels, respectively (r = 0.864 and r = 0.615, P < 0.01). Both the histoscores and mRNA levels of ephrinB2 and EphB4 significantly increased with clinical stages (I < II < III, P < 0.01), dedifferentiation (G(1) < G(2) < G(3), P < 0.01) and myometrial invasion (A < B < C, P < 0.01 for ephrinB2 and P < 0.05 for EphB4) in uterine endometrial cancers. The 60-month survival rates of the 34 patients with high ephrinB2 and EphB4 expression were poor (59% and 62% respectively), while for the other 34 patients with low ephrinB2 and EphB4 expression, they were significantly higher (85% and 82%, respectively). CONCLUSIONS: EphrinB2 and EphB4 were overexpressed during the tumor advancement as dedifferentiation and myometrial invasion. Therefore, ephrinB2/EphB4 might work on tumor advancement and may be recognized as a novel prognostic indicator for uterine endometrial cancers.

Adult↗

PubMed2004

Cell adhesion to ephrinb2 is induced by EphB4 independently of its kinase activity.

Cell to cell interaction in bone marrow is crucial for differentiation of hematopoietic cells. We have shown that EphB4 receptor is expressed in erythroid progenitor and its activation accelerates erythroid differentiation. To elucidate the role of EphB4 activation in erythropoiesis, we analyzed effects of EphB4 on cell adhesive pathways. Cell adhesion with the extension of filopodial pseudopod was observed by EphB4 activation. EphB4 activation also enhanced an effect of fibronectin-mediated adhesive pathway along with formation of the c-Cbl/CrkL complex. The tyrosine kinase activity of EphB4 was dispensable for those phenomena. These results suggest that activation of EphB4 participates in adhesive but not repulsive signals independently of its tyrosine kinase activity in hematopoietic cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis.

Bone homeostasis requires a delicate balance between the activities of bone-resorbing osteoclasts and bone-forming osteoblasts. Various molecules coordinate osteoclast function with that of osteoblasts; however, molecules that mediate osteoclast-osteoblast interactions by simultaneous signal transduction in both cell types have not yet been identified. Here we show that osteoclasts express the NFATc1 target gene Efnb2 (encoding ephrinB2), while osteoblasts express the receptor EphB4, along with other ephrin-Eph family members. Using gain- and loss-of-function experiments, we demonstrate that reverse signaling through ephrinB2 into osteoclast precursors suppresses osteoclast differentiation by inhibiting the osteoclastogenic c-Fos-NFATc1 cascade. In addition, forward signaling through EphB4 into osteoblasts enhances osteogenic differentiation, and overexpression of EphB4 in osteoblasts increases bone mass in transgenic mice. These data demonstrate that ephrin-Eph bidirectional signaling links two major molecular mechanisms for cell differentiation--one in osteoclasts and the other in osteoblasts--thereby maintaining bone homeostasis.

Animals↗

PubMed2006

Boning up on ephrin signaling.

The activities of osteoclasts, which degrade bone, and osteoblasts, which form bone, are coordinated to maintain bone homeostasis. Zhao et al. (2006) now demonstrate bidirectional signaling between these two cell populations via the transmembrane ligand ephrinB2 expressed by osteoclasts and its receptor EphB4 expressed by osteoblasts. Such bidirectional signaling limits osteoclast activity while stimulating osteoblast differentiation.

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↗

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↗

PubMed2006

Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.

EphB4, a member of the largest family of receptor tyrosine kinases, is normally expressed on endothelial and neuronal cells. Although aberrant expression of EphB4 has been reported in several human tumors, including breast cancer, its functional significance is not understood. We report here that EphB4 is expressed in 7 of 12 (58%) human breast cancer specimens and 4 of 4 (100%) breast tumor cell lines examined. Overexpression of EphB4 in breast cancer cells was driven by gene amplification and by the erbB family of receptors via activation of Janus tyrosine kinase-signal transducers and activators of transcription and protein kinase B. The aberrantly expressed receptor was phosphorylated by its natural ligand, EphrinB2, and signaled via the protein kinase B pathway. Targeted knockdown of EphB4 expression by small interference RNA (and antisense oligodeoxynucleotides (ODNs)) led to dose-dependent reduction in cell survival, increased apoptosis, and sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Antisense ODN-mediated EphB4 knockdown resulted in reduced tumor growth in a murine tumor xenograft model. Antisense ODN-treated tumors were 72% smaller than control tumors at 6 weeks, with an 86% reduction in proliferating cells, 15-fold increase in apoptosis, and 44% reduction in tumor microvasculature. Our data indicate that biologically active EphB4 functions as a survival factor in breast cancer and is a novel target for therapy.

Animals↗

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↗

PubMed2005

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development.

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.

Cell Line↗

PubMed2000

Prognostic significance of EPHB6, EFNB2, and EFNB3 expressions in neuroblastoma.

BACKGROUND: EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. PROCEDURE: Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. RESULTS: High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). CONCLUSIONS: EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups.

Child↗

PubMed2001

Control of retinal growth and axon divergence at the chiasm: lessons from Xenopus.

Metamorphosis in frogs is a critical developmental process through which a tadpole changes into an adult froglet. Metamorphic changes include external morphological transformations as well as important changes in the wiring of sensory organs and central nervous system. This review aims to provide an overview on the events that occur in the visual system of metamorphosing amphibians and to discuss recent studies that provide new insight into the molecular mechanisms that control changes in the retinal growth pattern as well as the formation of new axonal pathways in the central nervous system. BioEssays 23:319-326, 2001.

Animals↗

PubMed2002

Formation and maintenance of distinctive cell patterns by coexpression of membrane-bound ligands and their receptors.

We show that graded or checkerboard-like cell patterns, and segmental domains along a body axis, can be generated by cell behaviors involving differences in intercellular repulsion. A membrane-bound signal transduction system mediating contact-dependent cell interactions includes membrane-bound ligands (ephrins) and their receptors with tyrosine-kinase activity (Eph proteins). These molecules mediate both repulsive and attractive interactions under bilateral threshold control, i.e., cells expressing the receptors adhere to a surface bearing a critical density of ligand reciprocal to the density of receptor but are repelled by a surface with other densities of ligand (Honda [1998] J. Theor. Biol. 192:235-246). We extend this model. General membrane-bound ligands (not always ephrins) and their receptors are presumably coexpressed in a single cell under bilateral threshold control. Computer simulations of cell pattern formation showed that when coexpression of the ligand and receptor is reciprocal, the cells self-organize into a pattern of segmental domains or a graded cell arrangement along the body axis. The latter process interprets positional information in terms of protein molecules. When coexpression of the two species of molecules is not always reciprocal, the cells generate various patterns including checkerboard and kagome (star) patterns. The case of separate expression of ligands and receptors in different cells is also examined. The mechanism of differences in cell repulsion is compared with the differential cell adhesion hypothesis, which has been used to explain cell sorting.

Animals↗

PubMed2001

Ephrins stimulate neurite outgrowth during early cortical neurogenesis.

The Eph receptor ligands, the ephrins, are membrane-bound molecules that play important roles in establishing intercellular communication after neurogenesis by regulating cell migration, axon pathfinding, and topographic mapping. In diverse systems, such as embryonic day 17.5 (E17.5) hippocampal and cortical neurons, repulsive/inhibitory mechanisms underlie these cellular effects. However, although ligand/receptor expression occurs far earlier, during brain neurogenesis, little is known about potential ephrin functions in initial process outgrowth. We have examined ligand/receptor expression in E13.5 cortex in vivo and in culture, using alkaline phosphatase (AP)-conjugated reagents and RNase protection assay. B ephrins are highly expressed, including B1, B2, and B3, whereas A ephrins exhibit low expression levels. In contrast, the Eph receptors demonstrate an opposite pattern, exhibiting high levels of Eph A3, A4, and A5 mRNA transcripts and low levels of the B-class receptors. To examine effects on neurite outgrowth, soluble ephrins were incubated with antihuman IgG antibody, producing oligomeric agonist complexes, and dried onto culture dishes. Unexpectedly, both ephrin A and B complexes increased process outgrowth: Seventy to eighty percent of neuronal precursors exhibited long neurites on ephrins, whereas only 5-10% of cells had neurites on IgG control substrates, indicating that ephrins stimulated neuritogenesis by early cortical neurons. These observations suggest that ephrin ligand/receptor systems play ontogenetic roles not previously considered, activating mechanisms other than cellular repulsion. Ephrin systems may induce initial process elaboration by early cortical neurons that is restricted at later stages by well-characterized repulsive signaling mechanisms.

Animals↗

PubMed2001

Screening for soluble expression of recombinant proteins in a 96-well format.

For future structural and functional genomics programs new tools will be required. The implementation of high-throughput (HTP) methods for protein production will be an essential element. Present HTP protein production developments in structural genomics are aimed at obtaining well-expressing and highly soluble proteins, which are preferred candidates for structure-function studies. Here, we describe a cheap and efficient procedure to identify well-expressing soluble proteins in Escherichia coli in a compact 96-well format. Reproducible lysis on filter plates, followed by a filtration step on 96-well filter plates, allows the efficient separation of inclusion bodies from the soluble fraction. In the following step a dot blot procedure using anti-RGS-His(4) antibody (Qiagen) to detect expression of recombinant His-tagged protein is applied allowing direct detection of the target protein in the soluble fraction. The method is well suited for automation and should be applicable to expression screening of most proteins and fusion domains to which specific antibodies are available.

Biotechnology↗

PubMed1997

Phosphorylation of recombinant N-syndecan (syndecan 3) core protein.

The cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans is punctuated by the presence of four regularly spaced tyrosine residues. In this report, we explore the possibility of whether the four tyrosine residues in the cytoplasmic domain of N-syndecan (Syndecan 3) are potential substrates for phosphorylation by a tyrosine kinase. Bacterially expressed elk kinase was used to phosphorylate a series of bacterially expressed N-syndecan fusion proteins. Our results clearly demonstrate that the tyrosine residues in the cytoplasmic domain of N-syndecan can be phosphorylated by a tyrosine-specific kinase, and that all four tyrosine residues are capable of being phosphorylated.

Amino Acid Sequence↗

PubMed1999

Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus.

IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors. Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified. IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain. In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes. The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding. The amino-terminus of Jak2 is necessary for association with IL-12R beta 2.

Amino Acid Substitution↗