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PubMed1999

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis.

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

Animals↗

PubMed2006

EphB2 regulates axonal growth at the midline in the developing auditory brainstem.

Eph receptors play important roles in axon guidance at the midline. In the auditory system, growth of axons across the midline is an important determinant of auditory function. The avian cochlear nucleus, n. magnocellularis (NM), makes bilateral projections to its target, n. laminaris (NL). We examined the time course of NM axon growth toward the midline, the expression of Eph proteins at the midline during this growth, and the effects of Eph receptor misexpression on axonal growth across the midline. We found that NM axons reach the midline at E4. At this age, EphB receptors are expressed at the ventral floor plate. Expression extends dorsally to the ventricular zone beginning at E5. NM axons thus grow across the midline at a time when EphB receptor expression levels are low. Overexpression of EphB2 at E2 resulted in misrouted axons that deflected away from transfected midline cells. This effect was observed when midline cells were transfected but not when NM cells alone were transfected, suggesting that EphB2 acts non-cell autonomously and through reverse signaling. These data suggest an inhibitory role for midline Eph receptors, in which low levels permit axon growth and subsequently high levels prohibit growth after axons have crossed the midline.

Animals↗

PubMed2006

Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).

Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios.

Amino Acid Sequence↗

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↗

PubMed2000

Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina.

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.

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↗

PubMed2004

Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer.

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.

Cell Line, Tumor↗

PubMed2001

In vivo tyrosine phosphorylation sites of activated ephrin-B1 and ephB2 from neural tissue.

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.

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

Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats.

The present study provides the first evidence that signaling occurs between B-ephrins and EphB receptors in the adult CNS in response to injury. Specifically, our combined histological and biochemical data indicate that two members of the B-class of ephrins and Eph receptors, ephrin-B2 and EphB2, are expressed by astrocytes and meningeal fibroblasts, respectively, in the adult spinal cord. In response to thoracic spinal cord transection lesions, ephrin-B2 and EphB2 protein levels exhibit an initial decrease (1 d after lesion), followed by a significant increase by day 14. Immunohistochemical data indicate that ephrin-B2 is expressed by reactive CNS astrocytes, and EphB2 is present on fibroblasts invading the lesion site from the adjacent meninges. During the first 3 d after injury, there is intermingling of ephrin-B2-expressing reactive astrocytes at the lesion surface with EphB2-containing fibroblasts that is concurrent with bidirectional activation (phosphorylation) of ephrin-B2 and EphB2. By 7 d, both cell types are establishing restricted cellular domains containing dense networks of cells and interweaving processes. This astroglial-meningeal fibroblast scar is fully developed by day 14 when there is strict segregation of ephrin-B2-expressing astrocytes from EphB2-positive meningeal fibroblasts. These morphological changes are concomitant with a simultaneous decrease in ephrin-B2 and EphB2 activation. These observations provide strong evidence that cell contact-mediated bidirectional signaling between ephrin-B2 on reactive astrocytes and EphB2 on meningeal fibroblasts is an early event in the cellular cascades that result in the development of the glial scar and the exclusion of meningeal fibroblasts from the injured spinal cord.

Animals↗

PubMed2001

Genomic structure and loss of heterozygosity of EPHB2 in colorectal cancer.

EphB2, a member of the Eph receptor protein-tyrosine kinase family, is overexpressed in several human gastrointestinal tumors. Furthermore, the EphB2 gene is localized at 1p35-p36.1, a frequently deleted region in colon and other cancers. So, despite its overexpression in some kind of tumors, we decided to study the possibility of involvement in the EphB2 gene (EPHB2) mutation in colon cancers, because some of the well known tumor suppressor genes (e.g. p53) is overexpressed (really accumulated) in tumors. Fifty colon tumor samples of matched with their respective normal tissues, were studied for mutation of the EPHB2. Analysis of the genomic structure of EphB2 and survey of all 16 exons revealed an infrequent polymorphism (intron 2) and mutation (intron 8). Another polymorphism in exon 6, localized at nucleotide 1359 (A-->G) was found to be rather frequent in Japanese and Chinese subjects, but very rare in Caucasians. Taking advantage of this polymorphism within EPHB2, we surveyed the loss of heterozygosity (LOH) status of this gene in Japanese colorectal tumors. Among the 50 samples analyzed, 24 were informative, and LOH was found in five of the15 (33.3%) informative rectal cancer cases. Mutation analysis covering all 16 exons in the remaining allele did not reveal any mutations. Thus, EPHB2 is not a classical tumor suppressor gene.

Aged↗

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↗

PubMed2006

[Current situation of researches on the molecule mechanism of hormone refractory prostate cancer].

Most cases of prostate cancer become hormone refractory after 12 to 18 months of androgen deprivation therapy. The etiology of the disease is thought to be multifactorial, associated with genetic, dietary, and environmental factors. The article reviews the current situation of researches at home and abroad on the molecule mechanism of hormone refractory. It expounds the influence of the androgen receptor and its genetic mutation, apoptosis and the gene changes of p53, p21, EphB2 on prostate cancer. It is hoped to be of some directive value for the studies of prostate cancer.

Androgens↗

PubMed2006

EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined functions in development, but their expression patterns and functions in many adult tissues are still largely unknown. We have detected substantial levels of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult mouse kidney by RT-PCR amplification. Immunolocalization experiments revealed that EphB2 is localized in the tubules of the inner and outer medulla and EphB6 is in the tubules of the outer medulla and cortex. By contrast, ephrin-B1 was detected in tubules throughout the whole nephron. Consistent with the overlapping expression of the EphB2 receptor and the ephrin-B1 ligand in the medulla, EphB2 is tyrosine-phosphorylated, and therefore activated, in the kidney. In the outer medulla, however, EphB2 signaling may be attenuated by the co-expressed kinase-inactive EphB6 receptor. Interestingly, we found that EphB signaling induces RhoA activation and Rac1 inactivation as well as cell retraction, enlargement of focal adhesions and prominent stress fibers in primary cultures of medullary tubule cells. These results suggest that EphB receptor signaling through Rho family GTPases regulates the cytoarchitecture and spatial organization of the tubule cells in the adult kidney medulla and, therefore, may affect the reabsorption ability of the kidney.

Animals↗

PubMed2005

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1.

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 - a phosphatidylinositol 5'-phosphatase that is involved in clathrin-mediated endocytosis - as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5'-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB-EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Metastases and their microenvironments: linking pathogenesis and therapy.

The pathogenesis of metastasis depends on multiple favorable interactions of tumor cells with host homeostatic mechanisms. Interruption of one or more of these interactions can lead to the inhibition or eradication of cancer metastases. For many years, all efforts to treat cancer concentrated on the inhibition of growth or the destruction of tumor cells. A strategy of both eradication of tumor cells (e.g. by chemotherapy and immunotherapy) and modulation of the host microenvironment (e.g. tumor vasculature and hypoxia) is an additional, relatively novel approach to cancer treatment. Recent advances in our understanding of the biological basis of cancer metastasis open up unprecedented opportunities for translating basic research to clinical treatment of cancer. This research includes the unraveling of the genetic make-up of tumors and genome-wide expression analyses, thereby identifying many potential targets for therapy. Drugs acting on tumor cells which have a metastasis-prone mutational or expression status (by classical or targeted chemotherapy) as well as drugs affecting host-mediated survival pathways must be combined in order to create therapeutic synergy. Therapeutic maneuvers may target receptor tyrosine kinases (EGFR, VEGFR, FGFR), chemokines or G-protein-coupled receptors (CXCR4, CXCR2, EphB2), hypoxia-inducible factor (HIF), and signaling pathways (c-Src, PI3K, Akt, chaperon complexes) in tumor cells. Moreover, stromal and immunological cells and their cytokines coordinate critical pathways that exert important roles in the ability of tumors to invade and metastasize, thus suppressive cytokines (IL-6 and IL-10) and neutralizing specific antibodies might subvert conditions for metastasis.

Animals↗

PubMed2006

Intracellular and trans-synaptic regulation of glutamatergic synaptogenesis by EphB receptors.

The majority of mature excitatory synapses in the CNS are found on dendritic spines and contain AMPA- and NMDA-type glutamate receptors apposed to presynaptic specializations. EphB receptor tyrosine kinase signaling has been implicated in both NMDA-type glutamate receptor clustering and dendritic spine formation, but it remains unclear whether EphB plays a broader role in presynaptic and postsynaptic development. Here, we find that EphB2 is involved in organizing excitatory synapses through the independent activities of particular EphB2 protein domains. We demonstrate that EphB2 controls AMPA-type glutamate receptor localization through PDZ (postsynaptic density-95/Discs large/zona occludens-1) binding domain interactions and triggers presynaptic differentiation via its ephrin binding domain. Knockdown of EphB2 in dissociated neurons results in decreased functional synaptic inputs, spines, and presynaptic specializations. Mice lacking EphB1-EphB3 have reduced numbers of synapses, and defects are rescued with postnatal reexpression of EphB2 in single neurons in brain slice. These results demonstrate that EphB2 acts to control the organization of specific classes of mature glutamatergic synapses.

Animals↗

PubMed1999

Coexpression of transcripts encoding EPHB receptor protein tyrosine kinases and their ephrin-B ligands in human small cell lung carcinoma.

The EPH family is the largest subfamily of receptor protein tyrosine kinases, consisting of the EPHA and EPHB subgroups. Ephrin-B1, ephrin-B2, and ephrin-B3 are ligands of the EPHB subgroup and are encoded by the EFNB1, EFNB2, and EFNB3 genes, respectively. We have shown previously that EPHB2 transcripts are expressed in six small cell lung carcinoma (SCLC) cell lines. In this study, we examined the expression of EPHB1, EPHB2, EPHB3, EPHB4, and EPHB6 in 4 SCLC tumor specimens and 14 cell lines including 3 cell lines derived from these tumor specimens. To investigate whether potential autocrine loops of EPHB receptors and ephrin-B ligands exist in SCLC, the expression of EFNB1, EFNB2, and EFNB3 was also examined. Our data show that transcripts encoding multiple members of the EPHB subgroup and the ephrin-B subgroup are coexpressed in SCLC cell lines and tumors. These results suggest that the EPHB subgroup receptor kinases may modulate the biological behavior of SCLC through autocrine and/or juxtacrine activation by ephrin-B ligands that are expressed in the same or neighboring cells.

Autocrine Communication↗