Temporally compartmentalized expression of ephrin-B2 during renal glomerular development.
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Glomerular development proceeds through the spatially ordered and sequential recruitment, proliferation, assembly, and differentiation of endothelial, mesangial, and epithelial progenitors. The molecular determinants of cell-cell recognition and targeting in this process have yet to be defined. The Eph/ephrin family of membrane receptors and counter-receptors are critical participants of developmental vascular assembly in extrarenal sites. Renal expression patterns of ephrin-B2 and EphB4 were investigated using mice expressing beta-galactosidase under control of ephrin-B2 or EphB4 promoters. The earliest glomerular expression of ephrin-B2 was identified in a subset of differentiating comma-stage glomerular epithelial cells (podocyte progenitors) adjacent to the vascular cleft where endothelial progenitors are subsequently recruited. Epithelial ephrin-B2 expression was accompanied by expression in endothelial and mesangial cells as capillary assembly progressed. At or near completion of glomerular maturation, epithelial ephrin-B2 expression was extinguished, with persistence in glomerular endothelial cells. Throughout development, one of several ephrin-B2 receptors, EphB4, was persistently and exclusively expressed in endothelial cells of venous structures. The findings show sequential ephrin-B2 expression across glomerular lineages, first in a distinct subset of podocyte progenitors and subsequently in endothelial cells of the developing glomerulus. Given targeting functions for Eph/ephrin family proteins, the findings suggest that ephrin-B2 expression marks podocyte progenitors at the site of vascular cleft formation, where expression may establish an "address" to which endothelial and mesangial progenitors are recruited. Thus, the present results suggest that ephrin-B2 and EphB interactions play an important role in glomerular microvascular assembly. (+info)
The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.
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Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function. (+info)
Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors.
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Protein-protein interactions and calcium entry through the N-methyl-d-aspartate (NMDA)-type glutamate receptor regulate synaptic development and plasticity in the central nervous system. The EphB receptor tyrosine kinases are localized at excitatory synapses where they cluster and associate with NMDA receptors. We identified a mechanism whereby EphBs modulate NMDA receptor function. EphrinB2 activation of EphB in primary cortical neurons potentiates NMDA receptor-dependent influx of calcium. Treatment of cells with ephrinB2 led to NMDA receptor tyrosine phosphorylation through activation of the Src family of tyrosine kinases. These ephrinB2-dependent events result in enhanced NMDA receptor-dependent gene expression. Our findings indicate that ephrinB2 stimulation of EphB modulates the functional consequences of NMDA receptor activation and suggest a mechanism whereby activity-independent and activity-dependent signals converge to regulate the development and remodeling of synaptic connections. (+info)
Cardiovascular ephrinB2 function is essential for embryonic angiogenesis.
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EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. We have generated mice in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. We find that such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells. (+info)
Coexpression of ephrin-Bs and their receptors in colon carcinoma.
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BACKGROUND: The erythropoietin-producing hepatoma amplified sequence (Eph) family is the largest subfamily of receptor tyrosine kinases (RTKs). The Ephs (receptors) bind to specific cell-bound ligands, called ephrins. The binding of this ligand-receptor system is dependent on cell-cell interactions. The ephrin-Eph system is important in embryologic development and differentiation of the nervous and vascular systems. In the current study, the authors hypothesized that ephrins may play a role in the growth and development of colon carcinoma and may be expressed differentially in normal and malignant colonic tissues. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern Blot analyses, and immunohistochemistry were used to examine 11 colon carcinoma cell lines and 20 human colon carcinoma specimens with adjacent uninvolved mucosa for the expression of EphB and ephrin-B family members. RESULTS: EphB2, EphB3, and EphB4 mRNA expression and ephrin-B2 mRNA expression was detected in all the cell lines and colon carcinoma specimens examined. Immunohistochemical analysis showed that ephrin-B2 had higher expression in the colon carcinoma specimens studied than in adjacent normal mucosa. Ephrin-B2 and EphB4 most frequently were expressed on the luminal surface of colon carcinoma epithelium. CONCLUSIONS: The results of the current suggest that the ephrin-Bs are expressed differentially in colon carcinoma and normal mucosa specimens and thus may play a role in the progression of colon carcinoma. Further studies are necessary to determine the functional role of ephrin-Bs in colon carcinoma angiogenesis and growth. (+info)
EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase.
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Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as "receptor-like" signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Here we show that Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. Our data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling. (+info)
Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells.
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BACKGROUND: Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells. METHODS: In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested. RESULTS: MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR. CONCLUSIONS: ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment. (+info)
EphB ligand, ephrinB2, suppresses the VEGF- and angiopoietin 1-induced Ras/mitogen-activated protein kinase pathway in venous endothelial cells.
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Interaction between ephrinB2 and EphB4 in endothelial cells at the arterial-venous capillary interface is critical for proper embryonic capillary morphogenesis. However, the intracellular downstream signaling of ephrinB2-EphB in vascular endothelial cells is unknown. This study examined the effect of ephrinB2-induced activation of EphB kinases on vascular endothelial growth factor (VEGF)- and angiopoietin-1 (Ang1)-induced Ras/mitogen-activated protein kinase (MAPK) signaling cascades in human umbilical vein endothelial cells (HUVECs). Reverse transcriptase-polymer chain reaction results showed that HUVECs expressed three kinds of EphB kinases known to bind to ephrinB2: EphB2, EphB3, and EphB4. EphrinB2 not only increased the phosphorylation of EphB2 and EphB4 in a time-dependent manner but also increased recruitment of p120-Ras-GTPase-activating protein (p120-RasGAP) to EphB2 and EphB4. Accordingly, ephrinB2 inhibited VEGF- and Ang1-induced Ras-MAPK activities, whereas ephrinB2 did not alter VEGF-induced Flk phosphorylation or Ang1-induced Tie2 phosphorylation. Furthermore, ephrinB2 suppressed VEGF- and Ang1-induced proliferation and/or migration, which are mediated mainly through Ras/MAPK signaling cascades. From these results, we propose that ephrinB2-EphB, signaling through Ras/MAPK cascade, may be critical for proper morphogenesis of capillary endothelium through the arrest of endothelial cell proliferation and migration at the arterial-venous interface. (+info)