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PubMed1995

Molecular cloning of a ligand for the EPH-related receptor protein-tyrosine kinase Htk.

Htk is a receptor protein-tyrosine kinase that is related to the EPH subfamily of tyrosine kinases. The receptor has a wide tissue distribution including expression in several myeloid hematopoietic cell lines. Using an Htk-Fc fusion protein, a protein ligand for this receptor was expression cloned from the murine kidney mesangial cell line SV40MES 13. The Htk ligand cDNA encodes a transmembrane protein of 336 amino acids. Binding competition experiments demonstrated a Kd of 535 pM for binding of Htk-Fc to the Htk ligand. Incubation of 3T3 cells expressing Htk with COS-7 cells expressing the ligand resulted in tyrosine phosphorylation of Htk. The ligand, like its receptor, is widely expressed and may function in a variety of tissues. However, we localized hematopoietic expression of Htk to the monocytic lineage, suggesting that the ligand may play a role in differentiation and/or proliferation of these cells.

3T3 Cells↗

PubMed2002

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway.

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.

Alkaloids↗

PubMed2005

PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, we generated two sets of knock-in mice: ephrinB2DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2(5F) mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. Our analysis revealed that the homozygous mutant mice survived the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2DeltaV/DeltaV mice exhibited major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2(5F/5F) mice displayed only a mild lymphatic phenotype. Our studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions.

Amino Acid Sequence↗

PubMedDate not supplied

Expression of ephrin in retinal neovascularization and iris rubeosis.

We investigated expression of ephrin-B2 and Eph-B4 in the retinal tissues of six primate eyes with neovascularization and iris rubeosis secondary to laser-induced central retinal vein occlusion and in tissue from 10 human eyes with proliferative diabetic retinopathy. Two primate eyes with rubeosis and retinal neovascularization were enucleated 1, 2 and 4 weeks after the creation of central retinal vein occlusion. Antibodies were localized using the avidin-biotin reaction. In the primate eyes, ephrin-B2 was negative at I week and positive at 2 and 4 weeks in the rubeotic tissue, but was positive only at 2 weeks in the retinal neovascular membrane. Eph-B4 was negative in all the primate eye specimens. In the human tissue, ephrin-B2 was detected in two of the five eyes with rubeosis and three of the five eyes with retinal neovascularization. These data suggest that ephrin-B2 is a key regulator of neovascularization.

Angiogenic Proteins↗

PubMed1999

Expression of receptor tyrosine kinase HTK (hepatoma transmembrane kinase) and HTK ligand by human leukemia-lymphoma cell lines.

HTK (hepatoma transmembrane kinase) is a receptor tyrosine kinase belonging to the EPH subfamily of tyrosine kinases. Binding of its ligand (HTKL) results in tyrosine phosphorylation of HTK. In the present study, we analyzed the possible involvement of this ligand-receptor signalling system in hematopoiesis by examining the expression of both HTK and HTKL in a large and comprehensive panel of 70 continuous human leukemia-lymphoma cell lines. HTK and HTKL mRNA expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). HTK mRNA was detected in 68/70 cell lines; 58/70 cell lines were positive for HTKL mRNA expression; consequently, co-expression of both receptor and ligand was demonstrated in the majority of cell lines. Collectively, the wide-spread expression suggests a role for this ligand-receptor pair in hematopoietic development and/or function. Investigation of the details of signal transduction pathway that is activated by the HTK tyrosine kinase will help to define the exact biological function of the HTK-HTKL system.

Cell Line↗

PubMed1995

Cloning, characterization, and differential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of the eck/eph family.

Using a polymerase chain reaction-based strategy for the cloning of developmentally regulated receptor tyrosine kinases, we identified two novel members of the eck/eph-related subfamily which, in analogy with the recently identified mouse developmental kinase 1 (MDK1), were designated MDK2 and MDK5. MDK2 is highly homologous to the mouse kinase Myk-1 and the human kinase Htk, whereas MDK5 represents the mouse homologue of human Hek2. Northern blot analyses of adult mouse tissues revealed a 4.7 kb transcript of MDK2 and a 4.8 kb transcript of MDK5 in various organ systems, including lung, liver, kidney, intestine, muscle, heart, and, in the case of MDK5, also the brain. In addition to the full-length transcripts, smaller fragments were identified that probably represent truncated receptors. Northern blot analysis and in situ hybridization of mouse embryos indicated abundant expression during embryonic development, with preferential involvement of tissues of epithelial and endothelial origin for both kinases and of the spinal cord gray matter for MDK5. Unlike most other members of the eck/eph-related subfamily, the expression of MDK2 and MDK5 is not primarily restricted to neuronal structures, and their abundant presence in various organ systems during embryonic development suggests an important role in gestational growth and differentiation.

Amino Acid Sequence↗

PubMed1994

Expression of two novel eph-related receptor protein tyrosine kinases in mammary gland development and carcinogenesis.

To investigate the involvement of protein tyrosine kinases (PTKs) in the growth control of mammary epithelial cells, we have used PCR based cloning to identify PTKs expressed in a mouse mammary epithelial cell line. This approach led to the isolation of two receptor PTKs of the eph-related subfamily; myk-1, a novel member expressed predominantly in lung, heart and mammary gland and myk-2, a close relative of the human eck gene. Northern blot analysis of RNA from mouse mammary glands at different stages of development revealed that myk-1 and myk-2 expression is induced at puberty and differentially regulated during the estrus cycle. myk-1 and myk-2 expression was down-regulated during the pregnancy induced differentiation of the mammary gland. Over-expression of myk-1 and myk-2 was found in the undifferentiated and invasive mammary tumors of transgenic mice expressing the Ha-ras oncogene. In contrast, no elevated expression of either gene could be detected in the well differentiated and non-metastatic mammary tumors of c-myc expressing transgenic mice. These results indicate that myk-1 and myk-2 expression is induced during the proliferation of the mammary gland and down-regulated by its differentiation.

3T3 Cells↗

PubMed1994

Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily.

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

3T3 Cells↗

PubMed1996

Characterization of a ligand for receptor protein-tyrosine kinase HTK expressed in immature hematopoietic cells.

HTK is a receptor tyrosine kinase that belongs to the Eph subfamily. An extensive screening using BIAcore system revealed that a colon cancer cell line, C-1, expressed the ligand for HTK. From the conditioned medium of C-1 cells, a soluble form of ligand was purified by receptor affinity chromatography, and the isolation of full-length cDNA revealed that this ligand is identical to the human HTK ligand (HTKL) previously reported. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble HTKL but not by unclustered soluble HTKL, indicating that HTKL requires cell-to-cell interaction for receptor activation. Binding analysis demonstrated that HTKL binds to HTK with a much higher affinity (Kd: 1.23 nM) than the other transmembrane-type ligand for Eph family, LERK-2/ELKL (Kd: 135 nM). The expression of HTK in cord blood cells was upregulated after the culture in the presence of stem cell factor. Clustered soluble HTKL stimulated the proliferation of sorted HTK+ cord blood cells and a hematopoietic cell line, UT-7/EPO from which HTK was isolated. These findings suggest the involvement of HTK-HTKL system in the proliferation of HTK+ hematopoietic progenitor cells in the hematopoietic environment.

Amino Acid Sequence↗

PubMed1997

Selective expression of the receptor tyrosine kinase, HTK, on human erythroid progenitor cells.

HTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34(low), and glycophorin A(-/low). Assays of progenitors showed that HTK+ c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK- c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

Antibodies, Monoclonal↗

PubMed2005

Up-regulation of EphB4 in mesothelioma and its biological significance.

PURPOSE: Mesothelioma is a rare malignancy that is incurable and carries a short survival despite surgery, radiation, or chemotherapy. This study was designed to identify novel targets for diagnostic, prognostic, and therapeutic approaches. EXPERIMENTAL DESIGN: The expression and functional significance of the receptor tyrosine kinase EphB4 was studied in vitro and in a murine model of mesothelioma. RESULTS: EphB4 was highly expressed in mesothelioma cell lines and primary tumor tissues but not in normal mesothelium. Knockdown of EphB4 using small interfering RNA and antisense oligodeoxynucleotide showed reduction in cell survival, migration, and invasion. EphB4 knockdown initiated a caspase-8-mediated apoptosis and down-regulation of the anti-apoptotic protein bcl-xl. EphB4 knockdown also resulted in reduced phosphorylation of Akt and down-regulation of matrix metalloproteinase-2 transcription. In addition, murine tumor xenograft studies using EphB4 oligodeoxynucleotides showed a marked reduction in tumor growth accompanied by a specific decline in EphB4 protein levels, reduced cell division, apoptosis in tumor tissue, and decreased microvascular density. CONCLUSIONS: EphB4 is expressed in mesothelioma, provides a survival advantage to tumor cells, and is therefore a potential novel therapeutic target.

Animals↗

PubMed2003

Expression of EphB4 in head and neck squamous cell carcinoma.

EphB4 is a receptor tyrosine kinase that is expressed on epithelial cells during fetal life. It is also expressed on some venous endothelial cells. We conducted a study of six men with primary squamous cell carcinoma of the head and neck (HNSCC) that had metastasized to the cervical lymph nodes. Our goal was to determine if EphB4 is aberrantly expressed in cases of HNSCC and to determine if there is a qualitative difference between the expression of EphB4 on primary and metastatic tumors and its expression on normal mucosa adjacent to primary tumors. From each patient, we obtained specimens of the primary tumor, the nodal metastasis, and the adjacent normal mucosa, and we performed immunocytochemistry on each. We observed EphB4 expression in all primary and metastatic tumors and no expression in the normal tissue. In each of the six patients, expression was greater in the metastatic tumor than in the primary tumor. We conclude that EphB4 is a novel target in the treatment of HNSCC.

Adult↗

PubMed2004

Expression of Ephb2 and Ephb4 in breast carcinoma.

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, play key roles in diverse biological processes. Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, EphB receptors and ephrinB ligands are later proved to be crucial regulators of vasculogenesis and embryogenesis. More studies indicate that Eph receptors are involved in angiogenesis and tumorigenesis. This study aimed to investigate the expression of EphB2 and EphB4 in breast carcinomas. Semiquantitative RT-PCR and immunohistochemistry were used to examine the expression patterns of EphB2 and EphB4. Clinicopathological and survival correlations were statistically analyzed in a series of 94 breast carcinomas, 9 normal specimens and 4 breast carcinoma cell lines. 1(1%), 16(17%), 29(31%), 48(51%) of the 94 tumors were negative, weak, moderate and strong EphB2 protein expression, respectively. 6(6%), 27(29%), 28(30%), 33(35%) of the tumors were negative, weak, moderate and strong EphB4 expression, respectively. Both EphB2 and EphB4 RTPCR products could be detected in all specimens. Increased EphB2 protein expression was negatively associated with overall survival, and there was a trend that increased EphB2 protein expression was correlated with shorter disease free survival, while EphB4 protein expression was associated with histological grade and stage. EphB4 membrane staining was increased with S phase fraction and associated with DNA aneuploidy. These findings indicate that both EphB2 and EphB4 are involved in the development of breast cancer and that both molecules could be potential predictive markers.

Aneuploidy↗

PubMed2004

Syndecan-1 up-regulated by ephrinB2/EphB4 plays dual roles in inflammatory angiogenesis.

EphrinB2 and EphB4, its cognate receptor, are important in the vascular development of the mouse embryo. Their roles in human inflammatory angiogenesis, however, are not well understood. By examining hyperinflammatory lesions, we saw that ephrinB2 was predominantly expressed in macrophage-like cells and EphB4 in small venules. Because macrophages usually transmigrate through postcapillary venules during inflammation, we wanted to explore the downstream effects of EphB4 after binding to ephrinB2. By using cDNA microarray technique and following reverse transcriptase-polymerase chain reaction (RT-PCR), we found that syntenin and syndecan-1 were up-regulated in EphB4-positive endothelial cells dose dependently and time dependently after stimulation with preclustered ephrinB2. In vitro, ephrinB2 suppressed the angiogenic effects of basic fibroblast growth factor (bFGF) on EphB4-positive endothelial cells, partially due to syndecan-1's competition with fibroblast growth factor receptor (FGFR) for bFGF. However, ephrinB2 exhibited angiogenic effects in vivo, possibly due to an inflammation-associated enzyme-heparanase. The enzymes could convert the inhibitory effect of ephrinB2 on EphB4-positive endothelial cells to an activating effect by removing poorly sulfated side chains of up-regulated syndecan-1 ectodomain. Depending on the presence of heparanases, the roles of syndecan-1 may be opposite in different physiological settings.

Animals↗

PubMed2005

EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity.

The Eph receptor tyrosine kinases are overexpressed in many pathologic tissues and have therefore emerged as promising drug target candidates. However, there are few molecules available that can selectively bind to a single Eph receptor and not other members of this large receptor family. Here we report the identification by phage display of peptides that bind selectively to different receptors of the EphB class, including EphB1, EphB2, and EphB4. Peptides with the same EphB receptor specificity compete with each other for binding, suggesting that they have partially overlapping binding sites. In addition, several of the peptides contain amino acid motifs found in the G-H loop of the ephrin-B ligands, which is the region that mediates high-affinity interaction with the EphB receptors. Consistent with targeting the ephrin-binding site, the higher affinity peptides antagonize ephrin binding to the EphB receptors. We also designed an optimized EphB4-binding peptide with affinity comparable with that of the natural ligand, ephrin-B2. These peptides should be useful as selective inhibitors of the pathological activities of EphB receptors and as targeting agents for imaging probes and therapeutic drugs.

Amino Acid Motifs↗

PubMed2004

Eph and ephrin expression in normal placental development and preeclampsia.

Eph receptors and their ephrin ligands play a fundamental role in embryogenesis. Their functions include cell targeting and angiogenesis. In placental development, trophoblasts migrate and invade maternal tissue and spiral arteries, where they play a role in both anchoring the placenta to the uterus and increasing blood flow to the developing fetus (interstitial and endovascular invasions). We investigated the cellular distribution and expression patterns of representative Eph and ephrin RNA and protein in an effort to identify the molecules involved in trophoblast migration during normal placental development and placental pathologies. We found ephrin-A1 expressed exclusively in the invasive extravillous trophoblast (EVT) cell lineage. We therefore proceeded to investigate ephrin-A1 in placental pathologies with defects in EVT invasion. In preeclampsia, where trophoblast invasion is shallow, we observed ephrin-A1 expression similar to normal placenta. Furthermore, in initial experiments on the deeply invading trophoblasts of placenta accreta, which lacks decidua, ephrin-A1 is found to be expressed highly in extravillous trophoblasts that have invaded the myometrium. In addition, we found the prototype ephrin-A1 receptor, EphA2, localized in several placental cell types. EphB4 and ephrin-B2 molecules, which have specific expression patterns during artery and vein development, respectively, were also expressed in the placenta. The cell specific distribution of ephrin-A1 suggests that it may play a role in targeting and migration of trophoblasts, and in the vascular remodeling induced by the invading extravillous trophoblasts. Failure of ephrin-A1 expression is unlikely to be the primary cause in defective migration of trophoblasts observed in preeclampsia. Specific roles for other Eph and ephrin proteins remain to be investigated.

Blotting, Northern↗

PubMed2001

Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization.

The transmembrane ligand ephrinB2 and its receptor tyrosine kinase EphB4 are molecular markers of embryonic arterial and venous endothelial cells, respectively, and are essential for angiogenesis. Here we show that expression of ephrinB2 persists in adult arteries where it extends into some of the smallest diameter microvessels, challenging the classical view that capillaries have neither arterial nor venous identity. EphrinB2 also identifies arterial microvessels in several settings of adult neovascularization, including tumor angiogenesis, contravening the dogma that tumor vessels arise exclusively from postcapillary venules. Unexpectedly, expression of ephrinB2 also defines a stable genetic difference between arterial and venous vascular smooth muscle cells. These observations argue for revisions of classical concepts of capillary identity and the topography of neovascularization. They also imply that ephrinB2 may be functionally important in neovascularization and in arterial smooth muscle, as well as in embryonic angiogenesis.

Animals↗

PubMed2005

Endothelial ephrinB2 is controlled by microenvironmental determinants and associates context-dependently with CD31.

OBJECTIVE: The EphB ligand ephrinB2 has been identified as a critical determinant of arterial endothelial differentiation and as a positive regulator of invading endothelial cells during angiogenesis. This study was aimed at identifying determinants of endothelial cell ephrinB2 expression. METHODS AND RESULTS: Arteriovenous asymmetrical endothelial cell ephrinB2 expression in vivo is lost on transfer into culture with aortic endothelial cells becoming partially ephrinB2-negative and saphenous vein endothelial cells becoming partially ephrinB2-positive. Contact with smooth muscle cells and angiogenic stimulation by vascular endothelial growth factor lead to an increased endothelial cell ephrinB2 expression. Quiescent, smooth muscle-contacting endothelial cells express ephrinB2 uniformly on their luminal surface. In contrast, monolayer endothelial cells translocate ephrinB2 to interendothelial cell junctions, which is strongly enhanced by EphB4-Fc-mediated receptor body activation. Junctional ephrinB2 colocalizes and coimmunoprecipitates with CD31. CONCLUSIONS: This study identifies distinct regulatory mechanisms of endothelial ephrinB2 expression and cellular distribution in quiescent and activated endothelial cells. The data demonstrate that endothelial cell ephrinB2 expression is controlled by microenvironmental determinants rather than being an intrinsic endothelial cell differentiation marker.

Aorta↗