EphB1 controls long-range cortical axon guidance through a cell non-autonomous role in GABAergic cells.

EphB1 is required for proper guidance of cortical axon projections during brain development, but how EphB1 regulates this process remains unclear. We show here that EphB1 conditional knockout (cKO) in GABAergic cells (Vgat-Cre), but not in cortical excitatory neurons (Emx1-Cre), reproduced the cortical axon guidance defects observed in global EphB1 KO mice. Interestingly, in EphB1 cKOVgat mice, the misguided axon bundles contained co-mingled striatal GABAergic and somatosensory cortical glutamatergic axons. In wild-type mice, somatosensory axons also co-fasciculated with striatal axons, notably in the globus pallidus, suggesting that a subset of glutamatergic cortical axons normally follows long-range GABAergic axons to reach their targets. Surprisingly, the ectopic axons in EphB1 KO mice were juxtaposed to major blood vessels. However, conditional loss of EphB1 in endothelial cells (Tie2-Cre) did not produce the axon guidance defects, suggesting that EphB1 in GABAergic neurons normally promotes avoidance of these ectopic axons from the developing brain vasculature. Together, our data reveal a new role for EphB1 in GABAergic neurons to influence proper cortical glutamatergic axon guidance during brain development.

Colorectal cancer-associated mutations impair EphB1 kinase function.

Erythropoietin-producing hepatoma (Eph) receptor tyrosine kinases regulate the migration and adhesion of cells that are required for many developmental processes and adult tissue homeostasis. In the intestinal epithelium, Eph signaling controls the positioning of cell types along the crypt-villus axis. Eph activity can suppress the progression of colorectal cancer (CRC). The most frequently mutated Eph receptor in metastatic CRC is EphB1. However, the functional effects of EphB1 mutations are mostly unknown. We expressed and purified the kinase domains of WT and five cancer-associated mutant EphB1 and developed assays to assess the functional effects of the mutations. Using purified proteins, we determined that CRC-associated mutations reduce the activity and stability of the folded structure of EphB1. By mammalian cell expression, we determined that CRC-associated mutant EphB1 receptors inhibit signal transducer and activator of transcription 3 and extracellular signal-regulated kinases 1 and 2 signaling. In contrast to the WT, the mutant EphB1 receptors are unable to suppress the migration of human CRC cells. The CRC-associated mutations also impair cell compartmentalization in an assay in which EphB1-expressing cells are cocultured with ligand (ephrin B1)-expressing cells. These results suggest that somatic mutations impair the kinase-dependent tumor suppressor function of EphB1 in CRC.

Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain.

Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.

Regulation of Connexin32 by ephrin receptors and T-cell protein-tyrosine phosphatase.

Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Connexin32 (Cx32) is a major gap junction protein in the liver and brain. Phosphorylation is integral to regulating connexin assembly, degradation, and electrical and metabolic coupling, as well as to interactions with molecular partners. Cx32 contains two intracellular tyrosine residues, and tyrosine phosphorylation of Cx32 has been detected after activation of the epidermal growth factor receptor; however, the specific tyrosine residue and the functional implication of this phosphorylation remain unknown. To address the limited available information on Cx32 regulation by tyrosine kinases, here we used the Cx32 C-terminal (CT) domain in an in vitro kinase-screening assay, which identified ephrin (Eph) receptor family members as tyrosine kinases that phosphorylate Cx32. We found that EphB1 and EphA1 phosphorylate the Cx32CT domain residue Tyr243 Unlike for Cx43, the tyrosine phosphorylation of the Cx32CT increased gap junction intercellular communication. We also demonstrated that T-cell protein-tyrosine phosphatase dephosphorylates pTyr243 The data presented above along with additional examples throughout the literature of gap junction regulation by kinases, indicate that one cannot extrapolate the effect of a kinase on one connexin to another.

Peripheral EphrinB1/EphB1 signalling attenuates muscle hyperalgesia in MPS patients and a rat model of taut band-associated persistent muscle pain.

BACKGROUND: Myofascial pain syndrome (MPS) is an important clinical condition that is characterized by chronic muscle pain and a myofascial trigger point (MTrP) located in a taut band (TB). Previous studies showed that EphrinB1 was involved in the regulation of pathological pain via EphB1 signalling, but whether EphrinB1-EphB1 plays a role in MTrP is not clear. METHODS: The present study analysed the levels of p-EphB1/p-EphB2/p-EphB3 in biopsies of MTrPs in the trapezius muscle of 11 MPS patients and seven healthy controls using a protein microarray kit. EphrinB1-Fc was injected intramuscularly to detect EphrinB1s/EphB1s signalling in peripheral sensitization. We applied a blunt strike to the left gastrocnemius muscles (GM) and eccentric exercise for 8 weeks with 4 weeks of recovery to analyse the function of EphrinB1/EphB1 in the muscle pain model. RESULTS: P-EphB1, p-EphB2, and p-EphB3 expression was highly increased in human muscles with MTrPs compared to healthy muscle. EphB1 (r = 0.723, n = 11, P < 0.05), EphB2 (r = 0.610, n = 11, P < 0.05), and EphB3 levels (r = 0.670, n = 11, P < 0.05) in the MPS group were significantly correlated with the numerical rating scale (NRS) in the MTrPs. Intramuscular injection of EphrinB1-Fc produces hyperalgesia, which can be partially prevented by pre-treatment with EphB1-Fc. The p-EphB1 contents in MTrPs of MPS animals were significantly higher than that among control animals (P < 0.01). Intramuscular administration of the EphB1 inhibitor EphB1-Fr significantly suppressed mechanical hyperalgesia. CONCLUSIONS: The present study showed that the increased expression of p-EphB1/p-EphB2/p-EphB3 was related to MTrPs in patients with MPS. This report is the first study to examine the function of EphrinB1-EphB1 signalling in primary muscle afferent neurons in MPS patients and a rat animal model. This pathway may be one of the most important and promising targets for MPS.

Neuronal GAP-Porf-2 transduces EphB1 signaling to brake axon growth.

Axonal outgrowth and guidance require numerous extracellular cues and intracellular mediators that transduce signals in the growth cone to regulate cytoskeletal dynamics. However, the way in which cytoskeletal effectors respond to these signals remains elusive. Here, we demonstrate that Porf-2, a neuron-expressed RhoGTPase-activating protein, plays an essential role in the inhibition of initial axon growth by restricting the expansion of the growth cone in a cell-autonomous manner. Furthermore, the EphB1 receptor is identified as an upstream controller that binds and regulates Porf-2 specifically upon extracellular ephrin-B stimulation. The activated EphB forward signal deactivates Rac1 through the GAP domain of Porf-2, which inhibits growth cone formation and brakes axon growth. Our results therefore provide a novel GAP that regulates axon growth and braking sequentially through Eph receptor-independent and Eph receptor-dependent pathways.

Sumoylation of EphB1 Suppresses Neuroblastoma Tumorigenesis via Inhibiting PKCγ Activation.

BACKGROUND/AIMS: An increasing number of studies have linked erythropoietin-producing hepatocellular carcinoma (Eph) family receptor tyrosine kinases to cancer progression. However, little knowledge is available about the regulation of their functions in cancer. METHODS: SUMOylation was analyzed by performing Ni2+-NTA pull-down assay and immunoprecipitation. Cell proliferation, anchorage-independent growth, and tumorigenesis in vivo were examined by cell counting kit-8, soft agar colony formation assay, and a xenograft tumor mouse model, respectively. RESULTS: We found that EphB1 was post-translationally modified by the small ubiquitin-like modifier (SUMO) protein at lysine residue 785. Analysis of wild-type EphB1 and SUMOylation-deficient EphB1 K785R mutant revealed that SUMOylation of EphB1 suppressed cell proliferation, anchorage-independent cell growth, and xenograft tumor growth. Mechanistic study showed that SUMOylation of EphB1 repressed activation of its downstream signaling molecule PKCγ, and consequently inhibited tumorigenesis. A reciprocal regulatory loop between PKCγ and SUMOylation of EphB1 was also characterized. CONCLUSION: Our findings identify SUMO1 as a novel key regulator of EphB1-mediated tumorigenesis.

Activated receptor tyrosine kinases in granulosa cells of ovulating follicles in mice.

Successful ovulation requires the actions of gonadotropins along with those mediated by growth factors binding to their receptor tyrosine kinases (RTKs). There are several growth factors such as epidermal growth factor family ligands and interleukins that play a role during ovulation initiated by the preovulatory surge of luteinizing hormone (LH). The aim of this project was to analyze growth factor signaling pathways induced by LH in mouse granulosa cells. Immature female mice were treated with equine chorionic gonadotropin (eCG) followed 48 hr later by human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. We performed protein array analysis where we identified higher phosphorylation of insulin-like growth factor 1 receptor (IGF1R), the fibroblast growth factor receptor 2 (FGFR2) and ephrin receptor B1 (EPHB1) in granulosa cells at 4 hr post-hCG compared to 0 hr hCG (p < 0.05). We report both a significant increase in transcript abundance (p < 0.05) and the phosphorylation level (p < 0.05) of the IGF1R in granulosa cells at hCG4h. The mRNA abundance of the Fgfr2 and Ephb1 receptors remained unaltered upon hCG treatment. Nonetheless, transcript abundance of the fibroblast growth factor 2 (Fgf2) ligand was elevated at hCG4h (p < 0.01). Based on these results we conclude that the preovulatory LH surge activates signaling pathways of IGF1R through increase in the expression of the Igf1r gene in granulosa cells of ovulating follicles in mice. The LH surge also appears to activate FGFR2 IIIc and EPHB1 signaling, although further investigation is required.

Intramembranous processing by γ-secretase regulates reverse signaling of ephrin-B2 in migration of microglia.

The Eph-ephrin system plays pivotal roles in cell adhesion and migration. The receptor-like functions of the ephrin ligands allow the regulation of intracellular processes via reverse signaling. γ-Secretase mediated processing of ephrin-B has previously been linked to activation of Src, a kinase crucial for focal adhesion and podosome phosphorylation. Here, we analyzed the role of γ-secretase in the stimulation of reverse ephrin-B2 signaling in the migration of mouse embryonic stem cell derived microglia. The proteolytic generation of the ephrin-B2 intracellular domain (ICD) by γ-secretase stimulates Src and focal adhesion kinase (FAK). Inhibition of γ-secretase decreased the phosphorylation of Src and FAK, and reduced cell motility. These effects were associated with enlargement of the podosomal surface. Interestingly, expression of ephrin-B2 ICD could rescue these effects, indicating that this proteolytic fragment mediates the activation of Src and FAK, and thereby regulates podosomal dynamics in microglial cells. Together, these results identify γ-secretase as well as ephrin-B2 as regulators of microglial migration.

Downstream mediators of Ten-m3 signalling in the developing visual pathway.

BACKGROUND: The formation of visuotopically-aligned projections in the brain is required for the generation of functional binocular circuits. The mechanisms which underlie this process are unknown. Ten-m3 is expressed in a broad high-ventral to low-dorsal gradient across the retina and in topographically-corresponding gradients in primary visual centres. Deletion of Ten-m3 causes profound disruption of binocular visual alignment and function. Surprisingly, one of the most apparent neuroanatomical changes-dramatic mismapping of ipsilateral, but not contralateral, retinal axons along the representation of the nasotemporal retinal axis-does not correlate well with Ten-m3's expression pattern, raising questions regarding mechanism. The aim of this study was to further our understanding of the molecular interactions which enable the formation of functional binocular visual circuits. METHODS: Anterograde tracing, gene expression studies and protein pull-down experiments were performed. Statistical significance was tested using a Kolmogorov-Smirnov test, pairwise-fixed random reallocation tests and univariate ANOVAs. RESULTS: We show that the ipsilateral retinal axons in Ten-m3 knockout mice are mismapped as a consequence of early axonal guidance defects. The aberrant invasion of the ventral-most region of the dorsal lateral geniculate nucleus by ipsilateral retinal axons in Ten-m3 knockouts suggested changes in the expression of other axonal guidance molecules, particularly members of the EphA-ephrinA family. We identified a consistent down-regulation of EphA7, but none of the other EphA-ephrinA genes tested, as well as an up-regulation of ipsilateral-determinants Zic2 and EphB1 in visual structures. We also found that Zic2 binds specifically to the intracellular domain of Ten-m3 in vitro. CONCLUSION: Our findings suggest that Zic2, EphB1 and EphA7 molecules may work as effectors of Ten-m3 signalling, acting together to enable the wiring of functional binocular visual circuits.

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

BACKGROUND: Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. OBJECTIVES: To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. METHODS: Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. RESULTS: Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. CONCLUSIONS: PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133+ /CD166+ /EphB1+ /EfnB2+ , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures.

Zic2 Controls the Migration of Specific Neuronal Populations in the Developing Forebrain.

Human mutations in ZIC2 have been identified in patients with holoprosencephaly and schizophrenia. Similarly, Zic2 mutant mice exhibit holoprosencephaly in homozygosis and behavioral and morphological schizophrenic phenotypes associated with forebrain defects in heterozygosis. Despite the devastating effects of mutations in Zic2, the cellular and molecular mechanisms that provoke Zic2-deficiency phenotypes are yet unclear. Here, we report a novel role for this transcription factor in the migration of three different types of forebrain neurons: the Cajal-Retzius cells that populate the surface of the telencephalic vesicles, an amygdaloid group of cells originated in the caudal pole of the telencephalic pallium, and a cell population that travels from the prethalamic neuroepithelium to the ventral lateral geniculate nucleus. Our results also suggest that the receptor EphB1, previously identified as a Zic2 target, may mediate, at least partially, Zic2-dependent migratory events. According to these results, we propose that deficiencies in cell motility and guidance contribute to most of the forebrain pathologies associated with Zic2 mutations. SIGNIFICANCE STATEMENT: Although the phenotype of Zic2 mutant individuals was reported more than 10 years ago, until now, the main function of this transcription factor during early development has not been precisely defined. Here, we reveal a previously unknown role for Zic2 in the migration of forebrain neurons such as Cajal-Retzius cells, interneurons moving to the ventral lateral geniculate nucleus, and neocortical cells going to the amygdala. We believe that the role of this transcription factor in certain populations of migratory cells contributes to defects in cortical layering and hypocellularity in the ventral LGN and amygdala and will contribute to our understanding of the devastating phenotypes associated with Zic2 mutations in both humans and mice.

GluA2 trafficking is involved in apoptosis of retinal ganglion cells induced by activation of EphB/EphrinB reverse signaling in a rat chronic ocular hypertension model.

EphB1, expressed in Müller cells, and ephrinB2, expressed in both Müller cells and retinal ganglion cells (RGCs), constitute an EphB/ephrinB reverse signaling in RGCs. Whether and how this reverse signaling is involved in RGC apoptosis in a rat chronic ocular hypertension (COH) model was investigated. In the COH model, both EphB1 and ephrinB2 were significantly increased and the reverse signaling was activated, which was accompanied by increased protein levels of phosphorylated (p) src, GluA2, and p-GluA2. Intravitreal injection of EphB2-Fc, an activator of ephrinB2, induced an increase in TUNEL-positive signals in normal retinae. A coimmunoprecipitation assay demonstrated direct interactions among ephrinB2, p-src, and GluA2. Moreover, in COH rats the expression of GluA2 proteins on the surface of retinal cells was decreased. Such GluA2 endocytosis could be prevented by preoperational intravitreal injection of 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo [3,4-d] pyrimidine (PP2), an inhibitor of src family tyrosine kinases, and possibly involved the protein interacting with C kinase 1 and phosphorylation of GluA2. In normal rats, intravitreal injection of EphB2-Fc caused changes in these protein levels similar to those observed in COH rats, which all could be avoided by preinjection of PP2. Patch-clamp experiments further showed that the current-voltage relationship of AMPA receptor-mediated EPSCs of RGCs exhibited stronger inward rectification in EphB2-Fc-injected rats. Furthermore, preinjection of PP2 or N-[3-[[4-[(3-aminopropyl)amino]butyl]amino]propyl]-1-naphthaleneacetamide trihydrochloride) (Naspm), a Ca(2+)-permeable GluA2-lacking AMPA receptor inhibitor, remarkably inhibited RGC apoptosis in either EphB2-Fc-injected or COH rats. Together, elevated GluA2 trafficking induced by activated EphB2/ephrinB2 reverse signaling likely contributes to RGC apoptosis in COH rats.

Molecular mechanisms controlling the migration of striatal interneurons.

In the developing telencephalon, the medial ganglionic eminence (MGE) generates many cortical and virtually all striatal interneurons. While the molecular mechanisms controlling the migration of interneurons to the cortex have been extensively studied, very little is known about the nature of the signals that guide interneurons to the striatum. Here we report that the allocation of MGE-derived interneurons in the developing striatum of the mouse relies on a combination of chemoattractive and chemorepulsive activities. Specifically, interneurons migrate toward the striatum in response to Nrg1/ErbB4 chemoattraction, and avoid migrating into the adjacent cortical territories by a repulsive activity mediated by EphB/ephrinB signaling. Our results also suggest that the responsiveness of MGE-derived striatal interneurons to these cues is at least in part controlled by the postmitotic activity of the transcription factor Nkx2-1. This study therefore reveals parallel mechanisms for the migration of MGE-derived interneurons to the striatum and the cerebral cortex.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor.

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

EphrinB/EphB signaling controls embryonic germ layer separation by contact-induced cell detachment.

BACKGROUND: The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells could in principle be sufficient to maintain germ layer integrity and prevent intermixing. However, in organisms as diverse as fly, fish, or amphibian, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment. PRINCIPAL FINDINGS: We show that localized detachment resulting from contact-induced signals at the boundary is at the core of ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors and activation of Rac and RhoA. CONCLUSION: This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum.

Tissue-specific venous expression of the EPH family receptor EphB1 in the skin vasculature.

BACKGROUND: The major arteries and veins are formed early during development. The molecular tools to identify arterial and venous endothelial cells improve our understanding of arterial-venous differentiation and branching morphogenesis. Compared with arterial differentiation, relatively little is known about what controls venous development, due to lack of definitive molecular markers for venous endothelial cells. RESULTS: Here we report that the antibody against EphB1, an EphB class receptor, makes it possible to establish a reliable whole-mount immunohistochemical analysis of venous identity with greater resolution than previously possible in embryonic and adult skin vasculature models. EphB1 expression is restricted to the entire venous vasculature throughout embryonic development to adulthood, whereas the previously established venous marker EphB4 is also detectable in lymphatic vasculature. This venous-restricted expression of EphB1 is established after the vascular remodeling of the primary capillary plexus has occurred. Compared with its venous-specific expression in the skin, however, EphB1 is not restricted to the venous vasculature in yolk sac, trunk and lung. CONCLUSIONS: These studies introduce EphB1 as a new venous-restricted marker in a tissue-specific and time-dependent manner.

Ephrin-B stimulation of calvarial bone formation.

INTRODUCTION: Ephrin-B2 on osteoclasts was reported to promote bone formation as part of homeostasis by activating the EphB4 tyrosine kinase receptor on osteoblasts. Little is known about the role of ephrin-B signaling to EphBs in developmental bone formation. RESULTS: We observed expression of an ephrin-B2 LacZ chimeric allele in the periosteum, sutural bone fronts, and dura mater of embryonic and neonatal mice. Expression in the adult skull was confined to sutures, but was heavily upregulated at sites of bone injury. Culture of embryonic calvariae with soluble recombinant ephrin-B2/Fc doubled their bone content without altering suture width or overall skull morphology. Ephrin-B2/Fc also stimulated osteoblast marker gene expression in cultured MC3T3 preosteoblastic cells without the need for type 1 collagen-induced differentiation. EphB4 was absent in embryonic and adult skulls. However, EphB1 and EphB2, both physiological receptors for ephrin-Bs, were expressed at sites of osteogenesis, and EphB1 knockout mice displayed a reduction in calvarial bone content compared to controls. CONCLUSIONS: These data support a role for ephrin-B2 in the development and healing of bone through activation of osteoblast-specific gene expression. EphB1 and EphB2 are likely candidates receptors for the ephrin-B2 in bone.

Co-expression of protein phosphatases in insect cells affects phosphorylation status and expression levels of proteins.

The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.