RESUMEN
Rab11 family-interacting protein 5 (Rab11fip5) is an adaptor protein that binds to the small GTPase Rab11, which has an important function in endosome recycling and trafficking of cellular proteins to the plasma membrane. Rab11fip5 is involved in many cellular processes, such as cytoskeleton rearrangement, iron uptake and exocytosis in neuroendocrine cells, and is also known as a candidate gene for autism-spectrum disorder. However, the role of Rab11fip5 during early embryonic development is not clearly understood. In this study, we identified Rab11fip5 as a protein that interacts with ephrinB1, a transmembrane ligand for Eph receptors. The PDZ binding motif in ephrinB1 and the Rab-binding domain in Rab11fip5 are necessary for their interaction in a complex. EphrinB1 and Rab11fip5 display overlapping expression in the telencephalon of developing amphibian embryos. The loss of Rab11fip5 function causes a reduction in telencephalon size and a decrease in the expression level of ephrinB1. Moreover, morpholino oligonucleotide-mediated knockdown of Rab11fip5 decreases cell proliferation in the telencephalon. The overexpression of ephrinB1 rescues these defects, suggesting that ephrinB1 recycling by the Rab11/Rab11fip5 complex is crucial for proper telencephalon development.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Efrina-B1/metabolismo , Telencéfalo/crecimiento & desarrollo , Telencéfalo/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proliferación Celular , Citoesqueleto , Endosomas/metabolismo , Efrina-B1/genética , Exocitosis , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Neurogénesis , Telencéfalo/citología , Xenopus laevisRESUMEN
Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.
Asunto(s)
Retinopatía Diabética , Efrina-B1 , Enfermedades de la Retina , Animales , Humanos , Ratones , Ratas , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Efrina-B1/genética , Efrina-B1/metabolismo , Glucosa/metabolismo , Mediadores de Inflamación/metabolismo , Retina/metabolismo , Enfermedades de la Retina/metabolismoRESUMEN
BACKGROUND: The apical surface (AS) of epithelial cells is highly specialized; it is important for morphogenetic processes that are essential to shape organs and tissues and it plays a role in morphogen and growth factor signaling. Apical progenitors in the mammalian neocortex are pseudoepithelial cells whose apical surface lines the ventricle. Whether changes in their apical surface sizes are important for cortical morphogenesis and/or other aspects of neocortex development has not been thoroughly addressed. RESULTS: Here we show that apical progenitors are heterogeneous with respect to their apical surface area. In Efnb1 mutants, the size of the apical surface is modified and this correlates with discrete alterations of tissue organization without impacting apical progenitors proliferation. CONCLUSIONS: Altogether, our data reveal heterogeneity in apical progenitors AS area in the developing neocortex and shows a role for Ephrin B1 in controlling AS size. Our study also indicates that changes in AS size do not have strong repercussion on apical progenitor behavior.
Asunto(s)
Neocórtex , Neuronas , Animales , Neuronas/metabolismo , Transducción de Señal , Efrina-B1/metabolismo , Mamíferos/metabolismoRESUMEN
Proopiomelanocortin (POMC) neurons are major regulators of energy balance and glucose homeostasis. In addition to being regulated by hormones and nutrients, POMC neurons are controlled by glutamatergic input originating from multiple brain regions. However, the factors involved in the formation of glutamatergic inputs and how they contribute to bodily functions remain largely unknown. Here, we show that during the development of glutamatergic inputs, POMC neurons exhibit enriched expression of the Efnb1 (EphrinB1) and Efnb2 (EphrinB2) genes, which are known to control excitatory synapse formation. In vivo loss of Efnb1 in POMC-expressing progenitors decreases the amount of glutamatergic inputs, associated with a reduced number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits and excitability of these cells. We found that mice lacking Efnb1 in POMC-expressing progenitors display impaired glucose tolerance due to blunted vagus nerve activity and decreased insulin secretion. However, despite reduced excitatory inputs, mice lacking Efnb2 in POMC-expressing progenitors showed no deregulation of insulin secretion and only mild alterations in feeding behavior and gluconeogenesis. Collectively, our data demonstrate the role of ephrins in controlling excitatory input amount into POMC-expressing progenitors and show an isotype-specific role of ephrins on the regulation of glucose homeostasis and feeding.
Asunto(s)
Efrina-B1/metabolismo , Glucosa/metabolismo , Proopiomelanocortina/metabolismo , Animales , Encéfalo/metabolismo , Metabolismo Energético/fisiología , Efrina-B1/fisiología , Efrina-B2/metabolismo , Efrina-B2/fisiología , Fármacos actuantes sobre Aminoácidos Excitadores/metabolismo , Homeostasis/fisiología , Masculino , Ratones , Ratones Noqueados , N-Metilaspartato/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder characterized by craniofacial, skeletal, and neurological anomalies and is caused by mutations in EFNB1. Heterozygous females are more severely affected by CFNS than hemizygous males, a phenomenon called cellular interference that results from EPHRIN-B1 mosaicism. In Efnb1 heterozygous mice, mosaicism for EPHRIN-B1 results in cell sorting and more severe phenotypes than Efnb1 hemizygous males, but how craniofacial dysmorphology arises from cell segregation is unknown and CFNS etiology therefore remains poorly understood. Here, we couple geometric morphometric techniques with temporal and spatial interrogation of embryonic cell segregation in mouse mutant models to elucidate mechanisms underlying CFNS pathogenesis. By generating EPHRIN-B1 mosaicism at different developmental timepoints and in specific cell populations, we find that EPHRIN-B1 regulates cell segregation independently in early neural development and later in craniofacial development, correlating with the emergence of quantitative differences in face shape. Whereas specific craniofacial shape changes are qualitatively similar in Efnb1 heterozygous and hemizygous mutant embryos, heterozygous embryos are quantitatively more severely affected, indicating that Efnb1 mosaicism exacerbates loss of function phenotypes rather than having a neomorphic effect. Notably, neural tissue-specific disruption of Efnb1 does not appear to contribute to CFNS craniofacial dysmorphology, but its disruption within neural crest cell-derived mesenchyme results in phenotypes very similar to widespread loss. EPHRIN-B1 can bind and signal with EPHB1, EPHB2, and EPHB3 receptor tyrosine kinases, but the signaling partner(s) relevant to CFNS are unknown. Geometric morphometric analysis of an allelic series of Ephb1; Ephb2; Ephb3 mutant embryos indicates that EPHB2 and EPHB3 are key receptors mediating Efnb1 hemizygous-like phenotypes, but the complete loss of EPHB1-3 does not fully recapitulate the severity of CFNS-like Efnb1 heterozygosity. Finally, by generating Efnb1+/Δ; Ephb1; Ephb2; Ephb3 quadruple knockout mice, we determine how modulating cumulative receptor activity influences cell segregation in craniofacial development and find that while EPHB2 and EPHB3 play an important role in craniofacial cell segregation, EPHB1 is more important for cell segregation in the brain; surprisingly, complete loss of EPHB1-EPHB3 does not completely abrogate cell segregation. Together, these data advance our understanding of the etiology and signaling interactions underlying CFNS dysmorphology.
Asunto(s)
Movimiento Celular/genética , Anomalías Craneofaciales/genética , Efrina-B1/genética , Cresta Neural/embriología , Cráneo/anomalías , Animales , Anomalías Craneofaciales/diagnóstico , Modelos Animales de Enfermedad , Embrión de Mamíferos , Desarrollo Embrionario/genética , Efrina-B1/metabolismo , Femenino , Heterocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Mosaicismo , Mutación , Cresta Neural/citología , Fenotipo , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Índice de Severidad de la Enfermedad , Factores Sexuales , Cráneo/embriología , Cromosoma X/genéticaRESUMEN
BACKGROUND: Variation in facial shape may arise from the combinatorial or overlapping actions of paralogous genes. Given its many members, and overlapping expression and functions, the EPH receptor family is a compelling candidate source of craniofacial morphological variation. We performed a detailed morphometric analysis of an allelic series of E14.5 Ephb1-3 receptor mutants to determine the effect of each paralogous receptor gene on craniofacial morphology. RESULTS: We found that Ephb1, Ephb2, and Ephb3 genotypes significantly influenced facial shape, but Ephb1 effects were weaker than Ephb2 and Ephb3 effects. Ephb2-/- and Ephb3-/- mutations affected similar aspects of facial morphology, but Ephb3-/- mutants had additional facial shape effects. Craniofacial differences across the allelic series were largely consistent with predicted additive genetic effects. However, we identified a potentially important nonadditive effect where Ephb1 mutants displayed different morphologies depending on the combination of other Ephb paralogs present, where Ephb1+/- , Ephb1-/- , and Ephb1-/- ; Ephb3-/- mutants exhibited a consistent deviation from their predicted facial shapes. CONCLUSIONS: This study provides a detailed assessment of the effects of Ephb receptor gene paralogs on E14.5 mouse facial morphology and demonstrates how the loss of specific receptors contributes to facial dysmorphology.
Asunto(s)
Efrina-B1 , Desarrollo Maxilofacial , Receptor EphB1 , Receptor EphB3 , Receptores de la Familia Eph , Animales , Efrina-B1/genética , Efrina-B1/metabolismo , Cara , Ratones , Mutación , Receptor EphB1/genética , Receptor EphB2/genética , Receptor EphB3/genética , Receptores de la Familia Eph/metabolismoRESUMEN
Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1-associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the nephrin-ephrin-B1 complex at the slit diaphragm. The nephrin-ephrin-B1-NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both in vitro analyses with human embryonic kidney 293 cells and in vivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1-NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of nephrin-ephrin-B1-NHERF2-ezrin-actin is a novel critical axis in the podocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.
Asunto(s)
Efrina-B1/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Podocitos/metabolismo , Podocitos/patología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Células HEK293 , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Ratones , Ratones Noqueados , Ratas , Ratas WistarRESUMEN
Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only â¼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.
Asunto(s)
Efrina-B1/metabolismo , Efrina-B2/metabolismo , Efrina-B3/metabolismo , Infecciones por Henipavirus/virología , Henipavirus/fisiología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/química , Animales , Fusión Celular , Efrina-B1/genética , Efrina-B2/genética , Efrina-B3/genética , Infecciones por Henipavirus/genética , Infecciones por Henipavirus/metabolismo , Humanos , Ratones , Mutación , Unión Proteica , Conformación Proteica , Receptores Virales/genética , Especificidad de la Especie , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
Astrocytes are implicated in synapse formation and elimination, which are associated with developmental refinements of neuronal circuits. Astrocyte dysfunctions are also linked to synapse pathologies associated with neurodevelopmental disorders and neurodegenerative diseases. Although several astrocyte-derived secreted factors are implicated in synaptogenesis, the role of contact-mediated glial-neuronal interactions in synapse formation and elimination during development is still unknown. In this study, we examined whether the loss or overexpression of the membrane-bound ephrin-B1 in astrocytes during postnatal day (P) 14-28 period would affect synapse formation and maturation in the developing hippocampus. We found enhanced excitation of CA1 pyramidal neurons in astrocyte-specific ephrin-B1 KO male mice, which coincided with a greater vGlut1/PSD95 colocalization, higher dendritic spine density, and enhanced evoked AMPAR and NMDAR EPSCs. In contrast, EPSCs were reduced in CA1 neurons neighboring ephrin-B1-overexpressing astrocytes. Overexpression of ephrin-B1 in astrocytes during P14-28 developmental period also facilitated evoked IPSCs in CA1 neurons, while evoked IPSCs and miniature IPSC amplitude were reduced following astrocytic ephrin-B1 loss. Lower numbers of parvalbumin-expressing cells and a reduction in the inhibitory VGAT/gephyrin-positive synaptic sites on CA1 neurons in the stratum pyramidale and stratum oriens layers of KO hippocampus may contribute to reduced inhibition and higher excitation. Finally, dysregulation of excitatory/inhibitory balance in KO male mice is most likely responsible for impaired sociability observed in these mice. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits.SIGNIFICANCE STATEMENT This report establishes a link between astrocytes and the development of excitatory and inhibitory balance in the mouse hippocampus during early postnatal development. We provide new evidence that astrocytic ephrin-B1 differentially regulates development of excitatory and inhibitory circuits in the hippocampus during early postnatal development using a multidisciplinary approach. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits and associated behaviors. Given widespread and growing interest in the astrocyte-mediated mechanisms that regulate synapse development, and the role of EphB receptors in neurodevelopmental disorders, these findings establish a foundation for future studies of astrocytes in clinically relevant conditions.
Asunto(s)
Astrocitos/metabolismo , Efrina-B1/metabolismo , Potenciales Postsinápticos Excitadores , Hipocampo/metabolismo , Potenciales Postsinápticos Inhibidores , Animales , Homólogo 4 de la Proteína Discs Large/metabolismo , Efrina-B1/genética , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Hipocampo/fisiología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Piramidales/metabolismo , Células Piramidales/fisiología , Conducta Social , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.
Asunto(s)
Glándulas Suprarrenales/enzimología , Catecolaminas/metabolismo , Células Cromafines/enzimología , Exocitosis , Receptor EphB6/metabolismo , Transducción de Señal , Glándulas Suprarrenales/citología , Animales , Catecolaminas/genética , Células Cromafines/citología , Efrina-B1/genética , Efrina-B1/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptor EphB6/genética , Caracteres Sexuales , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Quimerinas/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptor EphB2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Separación Celular , Proteínas Quimerinas/química , Efrina-B1/genética , Efrina-B1/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas , Proteínas Oncogénicas/química , Fosforilación , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/química , Receptor EphB2/química , Receptor EphB2/genética , Transducción de Señal , Dominios Homologos srcRESUMEN
Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/metabolismo , Efrina-B1/metabolismo , Glomérulos Renales/citología , Proteínas de la Membrana/metabolismo , Síndrome Nefrótico/patología , Podocitos/citología , Animales , Animales Recién Nacidos , Proteínas Portadoras/genética , Diafragma , Efrina-B1/genética , Células HEK293 , Humanos , Glomérulos Renales/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Síndrome Nefrótico/metabolismo , Fosforilación , Podocitos/metabolismo , Ratas , Ratas WistarRESUMEN
The formation of tissue boundaries is dependent on the cell-cell adhesion/repulsion system that is required for normal morphogenetic processes during development. The Smad ubiquitin regulatory factors (Smurfs) are E3 ubiquitin ligases with established roles in cell growth and differentiation, but whose roles in regulating cell adhesion and migration are just beginning to emerge. Here, we demonstrate that the Smurfs regulate tissue separation at mesoderm/ectoderm boundaries through antagonistic interactions with ephrinB1, an Eph receptor ligand that has a key role in regulating the separation of embryonic germ layers. EphrinB1 is targeted by Smurf2 for degradation; however, a Smurf1 interaction with ephrinB1 prevents the association with Smurf2 and precludes ephrinB1 from ubiquitination and degradation, since it is a substantially weaker substrate for Smurf1. Inhibition of Smurf1 expression in embryonic mesoderm results in loss of ephrinB1-mediated separation of this tissue from the ectoderm, which can be rescued by the coincident inhibition of Smurf2 expression. This system of differential interactions between Smurfs and ephrinB1 regulates the maintenance of tissue boundaries through the control of ephrinB protein levels.
Asunto(s)
Efrina-B1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Xenopus/genética , Xenopus/metabolismo , Animales , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Unión al GTP Monoméricas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans-endocytosis of the inter-cellular receptor-ligand EphB-ephrinB complex is required. The molecular mechanism underlying trans-endocytosis is poorly defined. Here we show that the process is clathrin- and Eps15R-mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co-cultures of EphB2- and ephrinB1-expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2-mediated cell repulsion as shown in a rescue experiment in the EphB2 co-culture assay where wild type Eps15R but not the clathrin-binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans-endocytosis and thereby cell repulsion.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Clatrina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Clatrina/química , Endocitosis , Efrina-B1/metabolismo , Células HeLa , Humanos , Ratones , Unión Proteica , Ratas , Receptor EphB2/metabolismoRESUMEN
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.
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Efrina-B1/metabolismo , Hiperalgesia/patología , Músculo Esquelético/patología , Mialgia/metabolismo , Síndromes del Dolor Miofascial/patología , Receptor EphB1/metabolismo , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Humanos , Hiperalgesia/complicaciones , Masculino , Células Musculares/metabolismo , Células Musculares/patología , Mialgia/complicaciones , Síndromes del Dolor Miofascial/complicaciones , Fosforilación , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Astrocyte-derived factors can control synapse formation and functions, making astrocytes an attractive target for regulating neuronal circuits and associated behaviors. Abnormal astrocyte-neuronal interactions are also implicated in neurodevelopmental disorders and neurodegenerative diseases associated with impaired learning and memory. However, little is known about astrocyte-mediated mechanisms that regulate learning and memory. Here, we propose astrocytic ephrin-B1 as a regulator of synaptogenesis in adult hippocampus and mouse learning behaviors. We found that astrocyte-specific ablation of ephrin-B1 in male mice triggers an increase in the density of immature dendritic spines and excitatory synaptic sites in the adult CA1 hippocampus. However, the prevalence of immature dendritic spines is associated with decreased evoked postsynaptic firing responses in CA1 pyramidal neurons, suggesting impaired maturation of these newly formed and potentially silent synapses or increased excitatory drive on the inhibitory neurons resulting in the overall decreased postsynaptic firing. Nevertheless, astrocyte-specific ephrin-B1 knock-out male mice exhibit normal acquisition of fear memory but enhanced contextual fear memory recall. In contrast, overexpression of astrocytic ephrin-B1 in the adult CA1 hippocampus leads to the loss of dendritic spines, reduced excitatory input, and impaired contextual memory retention. Our results suggest that astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and mediate excitatory synapse elimination through its interactions with neuronal EphB receptors. Indeed, a deletion of neuronal EphB receptors impairs the ability of astrocytes expressing functional ephrin-B1 to engulf synaptosomes in vitro Our findings demonstrate that astrocytic ephrin-B1 regulates long-term contextual memory by restricting new synapse formation in the adult hippocampus.SIGNIFICANCE STATEMENT These studies address a gap in our knowledge of astrocyte-mediated regulation of learning and memory by unveiling a new role for ephrin-B1 in astrocytes and elucidating new mechanisms by which astrocytes regulate learning. Our studies explore the mechanisms underlying astrocyte regulation of hippocampal circuit remodeling during learning using new genetic tools that target ephrin-B signaling in astrocytes in vivo On a subcellular level, astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and trigger astrocyte-mediated elimination of EphB receptor-containing synapses. Given the role EphB receptors play in neurodevelopmental disorders and neurodegenerative diseases, these findings establish a foundation for future studies of astrocyte-mediated synaptogenesis in clinically relevant conditions that can help to guide the development of clinical applications for a variety of neurological disorders.
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Astrocitos/metabolismo , Efrina-B1/metabolismo , Hipocampo/fisiología , Memoria/fisiología , Sinapsis/fisiología , Animales , Masculino , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiologíaRESUMEN
The study aims to investigate the analgesic effects of microRNA-129-5p (miR-129-5p) on bone cancer pain (BCP) by targeting Eph receptor B1 (EphB1) through the EphB1/EphrinB2 signaling pathway. BCP mice models were established, and C3H/HeJ female mice were classified into the normal, blank, negative control (NC), miR-129-5p mimics, miR-129-5p inhibitors, EphB1 knockout (KO), and miR-129-5p inhibitors + EphB1 KO groups. Quantitative reverse transcription polymerase chain reaction and Western blot analysis were used to evaluate the miR-129-5p expression, and messenger RNA (mRNA) and protein expressions of EphB1, p-EphB1, EphrinB2, and p-EphrinB2. EphB1 and EphrinB2 were highly activated in the tibias of BCP mice 7 days after the operation. EphB1 is a target gene of miR-129-5p. The mechanical withdrawal threshold increased in the miR-129-5p mimics, EphB1 KO and miR-129-5p inhibitors + EphB1 KO groups, but decreased in the miR-129-5p inhibitors group. Compared with the blank and the NC groups, the expression of miR-129-5p was significantly increased in the miR-129-5p mimics group, and the mRNA and protein expressions of EphrinB2, p-EphrinB2, EphB1, and p-EphB1 were significantly decreased, while in the miR-129-5p inhibitors group, the results were opposite (all P < 0.05); the mRNA and protein expressions of EphrinB2, p-EphrinB2, EphB1, and p-EphB1 were significantly decreased in the EphB1 KO group (all P < 0.05); the expression of miR-129-5p was significantly decreased in the miR-129-5p inhibitors + EphB1 KO group ( P < 0.05), while the mRNA and protein expressions of EphrinB2 and p-EphrinB2 were not significantly different ( P > 0.05). The results indicated that upregulated miR-129-5p alleviate BCP via downregulation of the EphB1/EphrinB2 signaling pathway.
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Neoplasias Óseas/complicaciones , Dolor en Cáncer/etiología , Dolor en Cáncer/genética , Efrina-B1/metabolismo , Efrina-B2/metabolismo , MicroARNs/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Dolor en Cáncer/fisiopatología , Efrina-B1/genética , Efrina-B2/genética , Femenino , Regulación de la Expresión Génica , Ratones , MicroARNs/genética , Umbral del Dolor , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tibia/diagnóstico por imagen , Tibia/patologíaRESUMEN
Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy.Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.
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Efrina-B1/genética , Efrina-B1/metabolismo , Proteínas de la Membrana/metabolismo , Nefrosis/metabolismo , Podocitos/metabolismo , Animales , Anticuerpos , Movimiento Celular , Células HEK293 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Nefrosis/inmunología , Fosforilación , Podocitos/patología , RatasRESUMEN
Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.
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Efrina-B1/metabolismo , Proteínas Inmovilizadas/metabolismo , Nanoestructuras/química , Polimetil Metacrilato/química , Receptor EphB2/metabolismo , Efrina-B1/química , Células HEK293 , Humanos , Proteínas Inmovilizadas/química , Ligandos , Nanoestructuras/ultraestructura , Agregado de Proteínas , Multimerización de Proteína , Receptor EphB2/químicaRESUMEN
Advanced solid tumors are exposed to hypoxic conditions over longer periods of time as they grow. Tumor hypoxia is a major factor that induces malignant progression, but most previous studies on tumor hypoxia were performed under short-term hypoxia for up to 72 hours and few studies have focused on tumor response to chronic hypoxic conditions. Here we show a molecular mechanism by which chronic hypoxia promotes invasive behavior in prostate cancer cells. We found that an epithelial-mesenchymal transition (EMT)-driving transcription factor, slug, is specifically upregulated under chronic hypoxia and promotes tumor cell migration and invasion. Unexpectedly, processes associated with EMT, such as loss of E-cadherin, are not observed under chronic hypoxia. Instead, expression of ephrin-B1, a ligand of Eph-related receptor tyrosine kinases, is markedly induced by slug through E-box motifs and promotes cell migration and invasion. Furthermore, slug and ephrin-B1 are highly coexpressed in chronic hypoxic cells of human prostate adenocarcinoma tissues after androgen deprivation, which is known to cause tumor hypoxia. Taken together, these results indicate that chronic hypoxia-induced slug promotes invasive behavior of prostate cancer cells by activating the expression of ephrin-B1. In addition, ephrin-B1 may be a novel therapeutic target in combination with androgen deprivation therapy for aggressive prostate cancer.