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Mark Henkemeyer - One of the best experts on this subject based on the ideXlab platform.
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sinusoidal Ephrin receptor ephb4 controls hematopoietic progenitor cell mobilization from bone marrow
Journal of Clinical Investigation, 2016Co-Authors: Hyeongil Kwak, Mark Henkemeyer, Ombretta Salvucci, Roberto Weigert, Jorge L Martineztorrecuadrada, Michael G Poulos, Jason M Butler, Giovanna TosatoAbstract:Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow. Stress signals from cancer and other conditions promote HSPC mobilization into circulation and subsequent homing to tissue microenvironments. HSPC infiltration into tissue microenvironments can influence disease progression; notably, in cancer, HSPCs encourage tumor growth. Here we have uncovered a mutually exclusive distribution of EPHB4 receptors in bone marrow sinusoids and Ephrin B2 ligands in hematopoietic cells. We determined that signaling interactions between EPHB4 and Ephrin B2 control HSPC mobilization from the bone marrow. In mice, blockade of the EPHB4/Ephrin B2 signaling pathway reduced mobilization of HSPCs and other myeloid cells to the circulation. EPHB4/Ephrin B2 blockade also reduced HSPC infiltration into tumors as well as tumor progression in murine models of melanoma and mammary cancer. These results identify EPHB4/Ephrin B2 signaling as critical to HSPC mobilization from bone marrow and provide a potential strategy for reducing cancer progression by targeting the bone marrow.
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Ephrin B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear
Developmental Biology, 2014Co-Authors: Steven Raft, Mark Henkemeyer, Leonardo R Andrade, Dongmei Shao, Haruhiko Akiyama, Doris K WuAbstract:Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand Ephrin-B2 (EfnB2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that EfnB2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of EfnB2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of EfnB2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the EfnB2 C-terminus caused abnormalities similar to those found in the conditional EfnB2 null homozygote. Analyses of fetal EfnB2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB–EfnB2 signaling-deficient mice.
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Ephrin B2 reverse signaling increases α5β1 integrin mediated fibronectin deposition and reduces distal lung compliance
American Journal of Respiratory Cell and Molecular Biology, 2013Co-Authors: Katherine M Bennett, Christopher Dravis, Maria D Afanador, Charitharth Vivek Lal, Elizabeth A Persad, Susan K Legan, George Chenaux, Michael T Dellinger, Rashmin C Savani, Mark HenkemeyerAbstract:Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand Ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator Ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of Ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the Ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of Ephrin-B2 reverse signaling in the lung was assessed in EfnB2LacZ/6YFΔV mutants that coexpress the intracellular truncated Ephrin-B2–β-galactosidase fusion and an intracellular point mutant Ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain–containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the α5 integrin receptor required for FN fibrillogenesis. These experiments indicate that Ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.
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ephB2 and Ephrin B2 regulate the ionic homeostasis of vestibular endolymph
Hearing Research, 2007Co-Authors: Christopher Dravis, Nobuhiko Yokoyama, Michael J Chumley, Shiniu Wei, Daniel C Marcus, Mark HenkemeyerAbstract:Abstract The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored Ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K+-secreting vestibular dark cells and Ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or Ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K+ and low in Na+. Direct measurement of this fluid in live animals revealed significant decreases in K+ concentration and endolymphatic potential in both EphB2 and Ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and Ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.
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bidirectional signaling mediated by Ephrin B2 and ephB2 controls urorectal development
Developmental Biology, 2004Co-Authors: Christopher Dravis, Nobuhiko Yokoyama, Michael J Chumley, Chad A Cowan, Robert Silvany, Jennifer Shay, Linda A Baker, Mark HenkemeyerAbstract:Abstract Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely Ephrin-B2 and the Ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking Ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and Ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and Ephrin molecules play an important role in these clinically significant midline cell–cell adhesion and fusion events.
Elena B. Pasquale - One of the best experts on this subject based on the ideXlab platform.
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PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity
2016Co-Authors: Roberta Noberini, Ombretta Salvucci, Giovanna Tosato, Sayantan Mitra, Fatima Valencia, Srinivas Duggineni, Ke Wei, Ziwei Huang, Elena B. PasqualeAbstract:The EphB4 receptor tyrosine kinase together with its preferred ligand, Ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the Ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits Ephrin-B2 binding. Although Ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-Ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo
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the ephb4 receptor promotes the growth of melanoma cells expressing the Ephrin B2 ligand
Pigment Cell & Melanoma Research, 2010Co-Authors: Elena B. Pasquale, Naiying Yang, Pablo Lopezbergami, James S Goydos, Dana Yip, Ameae M Walker, Iryna M EthellAbstract:Dear Sir, Cutaneous melanoma is the most aggressive form of skin cancer and several families of receptor tyrosine kinases have been implicated in its development and progression, including the Eph receptor family (Hess et al., 2007; Smalley et al., 2009). Among Eph receptors, EphA2 has been most extensively studied in melanoma and linked to increased malignancy (Hess et al., 2007; Margaryan et al., 2009). The roles of other Eph receptors in melanoma progression, however, have not been extensively characterized. A recent study has shown that overexpression of EphB4 in murine B16 melanoma cells (which do not express the preferred EphB4 ligand, Ephrin-B2) decreases the survival of Ephrin-B2-positive tumor endothelial cells, suggesting that EphB4 may function as a tumor suppressor in melanoma by inhibiting angiogenesis (Huang et al., 2007). However, the widespread expression EphB4 in human cancers (Pasquale, 2010), including melanomas (Figure S1), suggests a possible positive role in tumor progression. Our previous work showed that EphB4 promotes the migratory ability of a series of murine melanoma cell lines (Yang et al., 2006). SW1 and C19 represent distinctive clones derived from the same parental cell line. The more aggressive SW1 cells express high levels of EphB4 and have high migratory ability, whereas the less aggressive C19 cells have low EphB4 levels and migrate poorly. The two cell lines similarly express the Ephrin-B2 ligand. We previously reported that EphB4 endogenously co-expressed with Ephrin-B2 in SW1 cells or transiently transfected in C19 cells promotes cell migration in vitro by activating the RhoA GTPase, thus inducing actin cytoskeleton reorganization. Here we show that EphB4 co-expression with Ephrin-B2 also promotes SW1 and C19 cell growth in vitro and tumor growth in vivo. We generated 4 stable C19 clones overexpressing EphB4 (C19-EphB4) as well as SW1 and C19 clones expressing EGFP (SW1 and C19; Figures 1A and S2A) and examined their proliferation by measuring BrdU incorporation and apoptosis by Hoechst nuclear staining. SW1 cells, which express high levels of endogenous EphB4, proliferate faster and exhibit less apoptosis compared to C19 cells. The C19-EphB4 clones, where the transfected EphB4 is expressed at levels similar to those in the SW1 clones, have proliferation and apoptotic rates comparable to the SW1 cells (Figures 1B,C and S2B,C). These results show that coexpression of EphB4 with Ephrin-B2 promotes melanoma cell proliferation and survival in vitro. To investigate the in vivo role of EphB4 in melanoma growth, we used the stable clones to generate tumors in a mouse xenograft model. The SW1 and C19-EphB4 tumors grew faster than the C19 tumors over a period of 4.7 weeks, while there was no significant difference in body weight between the 3 groups of mice (Figure S2D). Enhancement of melanoma tumor growth by EphB4 was confirmed by injecting a mixture of 3 additional SW1, C19 or C19-EphB4 clones and measuring tumor growth for 7.5 weeks (Figure 1D). Several lung metastases were observed at 7.5 weeks in the mice bearing SW1 tumors, but not in the ones with either C19 or C19-EphB4 tumors (data not shown), suggesting that high EphB4 expression alone may not be sufficient to promote the formation of detectable metastases. Figure 1 EphB4 promotes melanoma cell proliferation and inhibits apoptosis in vitro and promotes melanoma tumor growth in vivo. (A) EphB4 expression levels in 3 SW1, C19 and C19-EphB4 clones (data for clone #1 are shown in Figure S2). Lysates were probed by immunoblotting ... Figure 2 EphB4 upregulates Erk and Akt phosphorylation and Bcl-2 expression, and promotes blood vessel enlargement in melanoma tumors. (A) EphB4 immunoprecipitates from tumors grown for 7.5 weeks were probed for phosphotyrosine (pTyr) and reprobed for EphB4. The ... To investigate the signaling pathways that promote the growth of melanoma cells expressing high EphB4 levels, we first assessed EphB4 tyrosine phosphorylation as an indication of receptor activation. This revealed that EphB4 is substantially activated in the SW1 and C19-EphB4 tumors (Figure 2A), consistent with the reported expression of the Ephrin-B2 ligand in both SW1 and C19 cells and the extensive cell-cell contacts present in the 3-dimensional tumor environment (Yang et al., 2006). We then examined the effects of EphB4 expression on the activation of the Erk1/2 and the Akt kinases, which are known to play a critical role in melanoma cell transformation, survival and proliferation (Gray-Schopfer et al., 2007; Lopez-Bergami et al., 2008). We detected significantly higher levels of Erk1/2 phosphorylation at threonine 202 and tyrosine 204 and Akt phosphorylation at serine 473 in SW1 and C19-EphB4 tumors compared to C19 tumors, indicating increased activation of Erk1/2 and Akt (Figure 2B,C). We also detected higher levels of the anti-apoptotic protein Bcl-2 in the SW1 and C19-EphB4 tumors than the C19 tumors (Figure 2D). The higher Erk1/2 and Akt activity (which are consistent with the EphB4-dependent activation of Akt previously observed in breast cancer and endothelial cells (Steinle et al., 2002; Kumar et al., 2006)) and the higher Bcl-2 expression, would all be expected to contribute to the faster growth of tumors expressing activated EphB4. Besides cell proliferation and apoptosis, angiogenesis is another important event that contributes to tumor progression. Given that EphB4 can activate reverse signaling through Ephrin-B2 on adjacent endothelial cells to promote angiogenesis and blood vessel remodeling (Pasquale, 2010), we also examined the effects of EphB4 on tumor vascularization. Quantitative analysis of CD31-stained tumor sections revealed that the blood vessels were significantly larger in both SW1 and C19-EphB4 tumors than in C19 tumors at both 4.7 weeks and 7.5 weeks (Figures 2E and S3A). No significant difference was observed in blood vessel densities between the 3 groups, although more blood vessels were formed over the same time period in the tumors expressing EphB4, given the larger size of these tumors. To verify that the vascular differences observed were not due to the different tumor sizes, we also examined tumors of similar size, which were collected at different times after melanoma cell injection. SW1 and C19-EphB4 tumors similar in size to the C19 tumors also had larger blood vessels (Fig. S3B). These data suggest that EphB4 expression causes blood vessel growth and enlargement in SW1 and C19-EphB4 melanoma tumors. If increased vascularization in EphB4-expressing tumors supports faster growth of tumor cells, this would result in larger tumors with similar vascular densities (Kerbel and Folkman, 2002). Although EphB4 promotes SW1 and C19 melanoma cell malignancy, this receptor has been reported to suppress tumorigenicity in breast and colorectal cancer cells (Pasquale, 2010). These divergent activities may depend in part on whether the Ephrin-B2 ligand is co-expressed and persistently activates the receptor and on other contextual factors. In addition, Ephrin-B2 can also transduce signals through its cytoplasmic domain, which are known as reverse signals and are triggered by binding to Eph receptors (Pasquale, 2010; Meyer et al., 2005). Since both EphB4 and Ephrin-B2 are present in SW1 and C19-EphB4 cells, we cannot exclude that some of the tumor promoting effects observed could be due to Ephrin-B2 reverse signaling. In conclusion, we show that besides promoting RhoA-dependent migration (Yang et al., 2006), EphB4 can promote the growth of melanomas expressing the Ephrin-B2 ligand by stimulating proliferation, survival and angiogenesis. Our findings suggest that upregulation of EphB4 receptor expression can play a role in melanoma progression, particularly in tumors where Erk and/or Akt are not highly activated by mutations.
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structural characterization of the epha4 Ephrin B2 complex reveals new features enabling eph Ephrin binding promiscuity
Journal of Biological Chemistry, 2010Co-Authors: Roberta Noberini, Xuelu Huan, Elena B. Pasquale, Jianxing SongAbstract:Abstract EphA and EphB receptors preferentially bind Ephrin-A and Ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all Ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with Ephrin-B2, which represents the first structure of an EphA-Ephrin-B interclass complex. A loose fit of the Ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln12 and Glu14 and Ephrin-B2. Mutation of Gln12 and Glu14 does not cause significant structural changes in EphA4 or changes in its affinity for Ephrin-A ligands. However, the EphA4 mutant has ∼10-fold reduced affinity for Ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass Ephrin binding. Thus, EphA4 uses different strategies to bind Ephrin-A or Ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln12 and Glu14 with Ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-Ephrin complexes.
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Ephrin independent regulation of cell substrate adhesion by the ephb4 receptor
Biochemical Journal, 2009Co-Authors: Nicole K Noren, Elena B. Pasquale, Naiying Yang, Morgan Silldorff, Ravichandra MutyalaAbstract:Receptor tyrosine kinases of the Eph family become tyrosine phosphorylated and initiate signalling events upon binding of their ligands, the Ephrins. Eph receptors such as EphA2 and EphB4 are highly expressed but poorly tyrosine phosphorylated in many types of cancer cells, suggesting a limited interaction with Ephrin ligands. Nevertheless, decreasing the expression of these receptors affects the malignant properties of cancer cells, suggesting that Eph receptors may influence cancer cells independently of Ephrin stimulation. Ligand-independent activities of Eph receptors in cancer, however, have not been demonstrated. By using siRNA (small interfering RNA) to downregulate EphB4 in MCF7 and MDA-MB-435 cancer cells, we found that EphB4 inhibits integrin-mediated cell substrate adhesion, spreading and migration, and reduces β1-integrin protein levels. Low expression of the EphB4 preferred ligand, Ephrin-B2, and minimal contact between cells in these assays suggest that cell contact-dependent stimulation of EphB4 by the transmembrane Ephrin-B2 ligand does not play a role in these effects. Indeed, inhibitors of Ephrin-B2 binding to endogenous EphB4 did not influence cell substrate adhesion. Increasing EphB4 expression by transient transfection inhibited cell substrate adhesion, and this effect was also independent of Ephrin stimulation because it was not affected by single amino acid mutations in EphB4 that impair Ephrin binding. The overexpressed EphB4 was tyrosine phosphorylated, and we found that EphB4 kinase activity is important for inhibition of integrin-mediated adhesion, although several EphB4 tyrosine phosphorylation sites are dispensable. These findings demonstrate that EphB4 can affect cancer cell behaviour in an Ephrin-independent manner.
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structure and thermodynamic characterization of the ephb4 Ephrin B2 antagonist peptide complex reveals the determinants for receptor specificity
Structure, 2006Co-Authors: Jill E Chrencik, Mitchell Koolpe, Alexei Brooun, Michael I Recht, Michelle L Kraus, Anand Kolatkar, Richard H Bruce, Georg Martinybaron, Hans Widmer, Elena B. PasqualeAbstract: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.
Nobuhiko Yokoyama - One of the best experts on this subject based on the ideXlab platform.
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ephB2 and Ephrin B2 regulate the ionic homeostasis of vestibular endolymph
Hearing Research, 2007Co-Authors: Christopher Dravis, Nobuhiko Yokoyama, Michael J Chumley, Shiniu Wei, Daniel C Marcus, Mark HenkemeyerAbstract:Abstract The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored Ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K+-secreting vestibular dark cells and Ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or Ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K+ and low in Na+. Direct measurement of this fluid in live animals revealed significant decreases in K+ concentration and endolymphatic potential in both EphB2 and Ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and Ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.
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bidirectional signaling mediated by Ephrin B2 and ephB2 controls urorectal development
Developmental Biology, 2004Co-Authors: Christopher Dravis, Nobuhiko Yokoyama, Michael J Chumley, Chad A Cowan, Robert Silvany, Jennifer Shay, Linda A Baker, Mark HenkemeyerAbstract:Abstract Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely Ephrin-B2 and the Ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking Ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and Ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and Ephrin molecules play an important role in these clinically significant midline cell–cell adhesion and fusion events.
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Ephrin B2 reverse signaling is required for axon pathfinding and cardiac valve formation but not early vascular development
Developmental Biology, 2004Co-Authors: Nobuhiko Yokoyama, Michael J Chumley, Chad A Cowan, Robert Silvany, Linda A Baker, Ankur Saxena, Deepak Srivastava, Mark HenkemeyerAbstract:Vascular development begins with the formation of a primary vascular plexus that is rapidly remodeled by angiogenesis into the interconnected branched patterns characteristic of mature vasculature. Several receptor tyrosine kinases and their ligands have been implicated to control early development of the vascular system. These include the vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) that bind VEGF, the Tie-1 and Tie-2 receptors that bind the angiopoietins, and the EphB4 receptor that binds the membrane-anchored ligand Ephrin-B2. Targeted mutations in the mouse germline have revealed essential functions for these molecules in vascular development. In particular, protein-null mutations that delete either EphB4 or Ephrin-B2 from the mouse have been shown to result in early embryonic lethality due to failed angiogenic remodeling. The venous expression of EphB4 and arterial expression of Ephrin-B2 has lead to the speculation that the interaction of these two molecules leads to bidirectional signaling into both the receptor-expressing cell and the ligand-expressing cell, and that both forward and reverse signals are required for proper development of blood vessels in the embryo. Indeed, targeted removal of the Ephrin-B2 carboxy-terminal cytoplasmic tail by another group was shown to perturb vascular development and result in the same early embryonic lethality as the null mutation, leading the authors to propose that Ephrin-B2 reverse signaling directs early angiogenic remodeling of the primary vascular plexus [Cell 104 (2001) 57]. However, we show here that the carboxy-terminal cytoplasmic domain of Ephrin-B2, and hence reverse signaling, is not required during early vascular development, but it is necessary for neonatal survival and functions later in cardiovascular development in the maturation of cardiac valve leaflets. We further show that Ephrin-B2 reverse signaling is required for the pathfinding of axons that form the posterior tract of the anterior commissure. Our results thus indicate that Ephrin-B2 functions in the early embryo as a typical instructive ligand to stimulate EphB4 receptor forward signaling during angiogenic remodeling and that later in embryonic development Ephrin-B2 functions as a receptor to transduce reverse signals involved in cardiac valve maturation and axon pathfinding.
Chad A Cowan - One of the best experts on this subject based on the ideXlab platform.
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bidirectional signaling mediated by Ephrin B2 and ephB2 controls urorectal development
Developmental Biology, 2004Co-Authors: Christopher Dravis, Nobuhiko Yokoyama, Michael J Chumley, Chad A Cowan, Robert Silvany, Jennifer Shay, Linda A Baker, Mark HenkemeyerAbstract:Abstract Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely Ephrin-B2 and the Ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking Ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and Ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and Ephrin molecules play an important role in these clinically significant midline cell–cell adhesion and fusion events.
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Ephrin B2 reverse signaling is required for axon pathfinding and cardiac valve formation but not early vascular development
Developmental Biology, 2004Co-Authors: Nobuhiko Yokoyama, Michael J Chumley, Chad A Cowan, Robert Silvany, Linda A Baker, Ankur Saxena, Deepak Srivastava, Mark HenkemeyerAbstract:Vascular development begins with the formation of a primary vascular plexus that is rapidly remodeled by angiogenesis into the interconnected branched patterns characteristic of mature vasculature. Several receptor tyrosine kinases and their ligands have been implicated to control early development of the vascular system. These include the vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) that bind VEGF, the Tie-1 and Tie-2 receptors that bind the angiopoietins, and the EphB4 receptor that binds the membrane-anchored ligand Ephrin-B2. Targeted mutations in the mouse germline have revealed essential functions for these molecules in vascular development. In particular, protein-null mutations that delete either EphB4 or Ephrin-B2 from the mouse have been shown to result in early embryonic lethality due to failed angiogenic remodeling. The venous expression of EphB4 and arterial expression of Ephrin-B2 has lead to the speculation that the interaction of these two molecules leads to bidirectional signaling into both the receptor-expressing cell and the ligand-expressing cell, and that both forward and reverse signals are required for proper development of blood vessels in the embryo. Indeed, targeted removal of the Ephrin-B2 carboxy-terminal cytoplasmic tail by another group was shown to perturb vascular development and result in the same early embryonic lethality as the null mutation, leading the authors to propose that Ephrin-B2 reverse signaling directs early angiogenic remodeling of the primary vascular plexus [Cell 104 (2001) 57]. However, we show here that the carboxy-terminal cytoplasmic domain of Ephrin-B2, and hence reverse signaling, is not required during early vascular development, but it is necessary for neonatal survival and functions later in cardiovascular development in the maturation of cardiac valve leaflets. We further show that Ephrin-B2 reverse signaling is required for the pathfinding of axons that form the posterior tract of the anterior commissure. Our results thus indicate that Ephrin-B2 functions in the early embryo as a typical instructive ligand to stimulate EphB4 receptor forward signaling during angiogenic remodeling and that later in embryonic development Ephrin-B2 functions as a receptor to transduce reverse signals involved in cardiac valve maturation and axon pathfinding.
David J Anderson - One of the best experts on this subject based on the ideXlab platform.
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symmetrical mutant phenotypes of the receptor ephb4 and its specific transmembrane ligand Ephrin B2 in cardiovascular development
Molecular Cell, 1999Co-Authors: Sebastian S Gerety, Hai U Wang, Zhoufeng Chen, David J AndersonAbstract:Ephrin-B2 is a transmembrane ligand that is specifically expressed on arteries but not veins and that is essential for cardiovascular development. However, Ephrin-B2 is also expressed in nonvascular tissues and interacts with multiple EphB class receptors expressed in both endothelial and nonendothelial cell types. Thus, the identity of the relevant receptor for Ephrin-B2 and the site(s) where these molecules interact to control angiogenesis were not clear. Here we show that EphB4, a specific receptor for Ephrin-B2, is exclusively expressed by vascular endothelial cells in embryos and is preferentially expressed on veins. A targeted mutation in EphB4 essentially phenocopies the mutation in Ephrin-B2. These data indicate that Ephrin-B2–EphB4 interactions are intrinsically required in vascular endothelial cells and are consistent with the idea that they mediate bidirectional signaling essential for angiogenesis.
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molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by Ephrin B2 and its receptor eph b4
Cell, 1998Co-Authors: Hai U Wang, Zhoufeng Chen, David J AndersonAbstract:The vertebrate circulatory system is composed of arteries and veins. The functional and pathological differences between these vessels have been assumed to reflect physiological differences such as oxygenation and blood pressure. Here we show that Ephrin-B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells from the onset of angiogenesis. Conversely, Eph-B4, a receptor for Ephrin-B2, marks veins but not arteries. Ephrin-B2 knockout mice display defects in angiogenesis by both arteries and veins in the capillary networks of the head and yolk sac as well as in myocardial trabeculation. These results provide evidence that differences between arteries and veins are in part genetically determined and suggest that reciprocal signaling between these two types of vessels is crucial for morphogenesis of the capillary beds.