RESUMEN
Ephrin type-A receptor 10 (EPHA10) has been implicated as a potential target for breast and prostate cancer therapy. However, its involvement in oral squamous cell carcinoma (OSCC) remains unclear. We demonstrated that EPHA10 supports in vivo tumor growth and lymphatic metastasis of OSCC cells. OSCC cell migration, epithelial mesenchymal transition (EMT), and sphere formation were found to be regulated by EPHA10, and EPHA10 was found to drive expression of some EMT- and stemness-associated transcription factors. Among EPHA10 ligands, exogenous ephrin A4 (EFNA4) induced the most OSCC cell migration and sphere formation, as well as up-regulation of SNAIL, NANOG, and OCT4. These effects were abolished by extracellular signal-regulated kinase (ERK) inhibition and NANOG knockdown. Also, EPHA10 was required for EFNA4-induced cell migration, sphere formation, and expression of NANOG and OCT4 mRNA. Our microarray dataset revealed that EFNA4 mRNA expression was associated with expression of NANOG and OCT4 mRNA, and OSCC patients showing high co-expression of EFNA4 with NANOG or OCT4 mRNA demonstrated poor recurrence-free survival rates. Targeting forward signaling of the EFNA4-EPHA10 axis may be a promising therapeutic approach for oral malignancies, and the combination of EFNA4 mRNA and downstream gene expression may be a useful prognostic biomarker for OSCC.
Asunto(s)
Movimiento Celular , Efrina-A4/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca/patología , Proteína Homeótica Nanog/metabolismo , Receptores de la Familia Eph/metabolismo , Esferoides Celulares/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Efrina-A4/genética , Transición Epitelial-Mesenquimal , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Proteína Homeótica Nanog/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Receptores de la Familia Eph/genética , Esferoides Celulares/metabolismo , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin (IL)-6 is a proinflammatory cytokine released in injured and contracting skeletal muscles. In this study, we examined cellular expression of proteins associated with cytoskeleton organization and cell migration, chosen on the basis of microRNA profiling, in rat primary skeletal muscle cells (RSkMC) treated with IL-6 (1 ng/ml) for 11 days. MiRNA microarray analysis and qRT-PCR revealed increased expression of miR-154-3p and miR-338-3p in muscle cells treated with IL-6. Pacsin3 was downregulated post-transcriptionally by IL-6, but not by IGF-I. Ephrin4A protein was increased both in IL-6- and IGF-I-treated myocytes. IL-6, but not IGF-I, stimulated migratory ability of RSkMC, examined in wound healing assay. Alpha-actinin protein was slightly augmented in RSKMC treated with IL-6, similarly to IGF-I. IL-6, but not IGF-I, upregulated desmin in differentiating RSkMC. IL-6 supplementation caused accumulation of alpha-actinin and desmin in near-nuclear area of muscle cells, which was manifested by increased ratio: mean near-nuclear fluorescence/mean peripheral cytoplasm fluorescence of these proteins. We concluded that IL-6, a known proinflammatory cytokine and a physical activity-associated myokine, acting during differentiation of primary skeletal muscle cells, alters expression of nonmuscle-specific miRNAs. This cytokine causes differential effects on pacsin-3 and ephrinA4, through post-transcriptional inhibition and stimulation, respectively. IL-6-exerted modifications of cytoskeletal proteins in muscle cells include both transcriptional (desmin and dynein heavy chain 5) and post-transcriptional activation (alpha-actinin). Moreover, IL-6 augments near-nuclear distribution of cytoskeletal proteins, alpha-actinin and desmin and promotes migration of myocytes. Such effects suggest that IL-6 plays a role during skeletal muscle regeneration, acting through mechanisms independent of regulation of myogenic program.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Efrina-A4/biosíntesis , Interleucina-6/farmacología , Mioblastos Esqueléticos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/efectos de los fármacos , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Efrina-A4/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , MicroARNs/biosíntesis , MicroARNs/genética , MicroARNs/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Procesamiento Postranscripcional del ARN , Ratas , Proteínas Recombinantes/farmacología , Transcripción GenéticaRESUMEN
Craniosynostosis (CS) has a prevalence of approximately 1 in every 2000 live births and is characterized by the premature fusion of one or more cranial sutures. Failure to maintain the cell lineage boundary at the coronal suture is thought to be involved in the pathology of some forms of CS. The Ephrin family of receptor tyrosine kinases consists of membrane-bound receptors and ligands that control cell patterning and the formation of developmental boundaries. Mutations in the ephrin A4 (EFNA4) and ephrin B1 (EFNB1) ligands have been linked to nonsyndromic CS and craniofrontonasal syndrome, respectively, in patient samples. We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if EFNA4 or EFNB1 could be the loci of the causal mutation in this unique animal model. Sequencing of EFNA4 and EFNB1 was performed using templates obtained from wild-type (n = 4) and craniosynostotic (n = 4) rabbits. No structural coding errors were identified in either gene. A single-nucleotide transversion was identified in one wild-type rabbit within the third intron of EFNA4. These data indicate that the causal locus for heritable CS in this rabbit model is not located within the structural coding regions of either EFNA4 or EFNB1.
Asunto(s)
Craneosinostosis/genética , Efrina-A4/genética , Efrina-B1/genética , Animales , Modelos Animales de Enfermedad , Intrones , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , ConejosRESUMEN
A role of endothelial cells in the survival of CLL cells during extravasation is presently unknown. Herein we show that CLL cells but not normal B cells can receive apoptotic signals through physical contact with TNF-α activated endothelium impairing survival in transendothelial migration (TEM) assays. In addition, the CLL cells of patients having lymphadenopathy (LApos) show a survival advantage during TEM that can be linked to increased expression of α4 and αL integrin chains. Within this context, ephrinA4 expressed on the surface of CLL cells sequestrates integrins and inactivates them resulting in reduced adhesion and inhibition of apoptotic/survival signals through them. In agreement, ephrinA4 silencing resulted in increased survival of CLL cells of LApos patients but not LA neg patients. Similarly was observed when a soluble ephrinA4 isoform was added to TEM assays strongly suggesting that accumulation of this isoform in the serum of LApos patients could contribute to CLL cells dissemination and survival in vivo. In supporting, CLL lymphadenopathies showed a preferential accumulation of apoptotic CLL cells around high endothelial venules lacking ephrinA4. Moreover, soluble ephrinA4 isolated from sera of patients increased the number and viability of CLL cells recovered from the lymph nodes of adoptively transferred mice. Finally, we present evidence suggesting that soluble ephrinA4 mediated survival during TEM could enhance a transcellular TEM route of the CLL cells. Together these findings point to an important role of ephrinA4 in the nodal dissemination of CLL cells governing extravasation and survival.
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Apoptosis , Antígeno CD11a/metabolismo , Supervivencia Celular , Efrina-A4/metabolismo , Integrina alfa4/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Anciano , Anciano de 80 o más Años , Animales , Linfocitos B/fisiología , Células Cultivadas , Técnicas de Cocultivo , Endotelio/metabolismo , Efrina-A4/sangre , Efrina-A4/genética , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Isoformas de Proteínas/sangre , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Organismos Libres de Patógenos Específicos , Migración Transendotelial y Transepitelial , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathologies conditions such as phobias and posttraumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). EphrinA4 and its cognate Eph receptors are intimately involved in regulating neuronal morphogenesis, synaptic transmission and plasticity. To assess possible roles of ephrinA4 in fear memory formation we designed and used a specific inhibitory ephrinA4 mimetic peptide (pep-ephrinA4) targeted to EphA binding site. We show that this peptide, composed of the ephrinA4 binding domain, interacts with EphA4 and inhibits ephrinA4-induced phosphorylation of EphA4. Microinjection of the pep-ephrinA4 into rat LA 30 min before training impaired long- but not short-term fear conditioning memory. Microinjection of a control peptide derived from a nonbinding E helix site of ephrinA4, that does not interact with EphA, had no effect on fear memory formation. Microinjection of pep-ephrinA4 into areas adjacent to the amygdala had no effect on fear memory. Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation. These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation. Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.
Asunto(s)
Amígdala del Cerebelo/fisiología , Efrina-A4/administración & dosificación , Miedo/fisiología , Memoria a Largo Plazo/fisiología , Animales , Sitios de Unión/genética , Condicionamiento Clásico/fisiología , Efrina-A4/genética , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo-A/Nogo receptor (NgR1) signaling pathway. Nogo-A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody-mediated neutralization of Nogo-A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo-A KO vs. wild-type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4-positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth-inhibitory than WT but more than Nogo-A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo-A KO myelin as compared with WT neurons, supporting increased EphA4-mediated growth inhibition in Nogo-A KO mice. Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo-A.
Asunto(s)
Axones/fisiología , Corteza Cerebral/metabolismo , Ganglios Espinales/metabolismo , Proteínas de la Mielina/metabolismo , Regeneración de la Medula Espinal , Regulación hacia Arriba , Animales , Axones/metabolismo , Células Cultivadas , Corteza Cerebral/patología , Corteza Cerebral/fisiología , Efrina-A3/genética , Efrina-A3/metabolismo , Efrina-A4/genética , Efrina-A4/metabolismo , Ganglios Espinales/patología , Ganglios Espinales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina/genética , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Proteínas Nogo , Tractos Piramidales/metabolismo , Tractos Piramidales/patología , Tractos Piramidales/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Traumatismos de la Médula Espinal/metabolismoRESUMEN
The Eph receptors represent the largest family of receptor tyrosine kinases. Both Eph receptors and their ephrin ligands are cell-surface proteins, and they typically mediate cell-to-cell communication by interacting at sites of intercellular contact. The major aim of the present study was to investigate the involvement of EphA4-ephrin-A1 interaction in monocyte adhesion to endothelial cells, as this process is a crucial step during the initiation and progression of the atherosclerotic plaque. Immunohistochemical analysis of human atherosclerotic plaques revealed expression of EphA4 receptor and ephrin-A1 ligand in major cell types within the plaque. Short-time stimulation of endothelial cells with the soluble ligand ephrin-A1 leads to a fourfold increase in adhesion of human monocytes to endothelial cells. In addition, ephrin-A1 further increases monocyte adhesion to already inflamed endothelial cells. EphrinA1 mediates its effect on monocyte adhesion via the activated receptor EphA4. This ephrinA1/EphA4 induced process involves the activation of the Rho signaling pathway and does not require active transcription. Rho activation downstream of EphA4 leads to increased polymerization of actin filaments in endothelial cells. This process was shown to be crucial for the proadhesive effect of ephrin-A1. The results of the present study show that ephrin-A1-induced EphA4 forward signaling promotes monocyte adhesion to endothelial cells via activation of RhoA and subsequent stress-fiber formation by a non-transcriptional mechanism.
Asunto(s)
Aterosclerosis/metabolismo , Adhesión Celular , Endotelio Vascular/metabolismo , Efrina-A1/metabolismo , Efrina-A4/metabolismo , Monocitos/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Western Blotting , Proliferación Celular , Células Cultivadas , Endotelio Vascular/citología , Efrina-A1/antagonistas & inhibidores , Efrina-A1/genética , Efrina-A4/antagonistas & inhibidores , Efrina-A4/genética , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
PURPOSE: Retinal neovascularization (NV) is a major cause of blindness. Recent research suggests that factors other than VEGF participate in this process. This study aimed to determine the role of ephrin-A4 in retinal NV. METHODS: The expression and effect of ephrin-A4 was investigated in a mouse model of oxygen-induced retinopathy (OIR) and the RF/6A retina endothelial cell line. Ephrin-A4 expression and VEGF signaling pathway phosphorylation were determined by PCR, immunohistochemistry, and western blot analyses. ShRNA was used to silence ephrin-A4 in vitro and in vivo. Retinal flat mounts and tube formation assays were performed to evaluate ephrin-A4 function in the NV process in vivo and in vitro. RESULTS: Ephrin-A4 was overexpressed in the retina of OIR mice and in RF/6A and RPE cells after CoCl2 stimulation. In vitro, Ephrin-A4/Fc treatment significantly increased the tube number of RF/6A cells on a membrane preparation and the phosphorylation levels of VEGR2, Akt1, and ERK1/2 in RF/6A cells. Moreover, ephrin-A4 knockout markedly suppressed pathologic neovascularization in vivo and inhibited the proliferation and tube formation capacity of RF/6A cells in vitro. Furthermore, in the absence of ephrin-A4, the phosphorylation of VEGFR2, Akt1, and ERK1/2 was defective under VEGF165 stimulation, and the proangiogenic function of VEGF165 was also compromised. CONCLUSIONS: This study suggests that ephrin-A4 plays an important role in retinal NV and is a potential target against retinal NV. The proangiogenic function of ephrin-A4 may be linked to its crucial role in the VEGF signaling pathway.
Asunto(s)
Efrina-A4/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Neovascularización Retiniana/metabolismo , Epitelio Pigmentado de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Efrina-A4/biosíntesis , Estudios de Seguimiento , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Neovascularización Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family -but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.
Asunto(s)
Retrovirus Endógenos/patogenicidad , Efrinas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Infecciones por Retroviridae/virología , Animales , Chlorocebus aethiops , Retrovirus Endógenos/genética , Efrina-A4/genética , Efrina-A4/metabolismo , Efrinas/genética , Femenino , Regulación Viral de la Expresión Génica , Biblioteca de Genes , Genes de Partícula A Intracisternal/genética , Genes Virales , Células HEK293 , Humanos , Ratones , Ovario/metabolismo , Infecciones por Retroviridae/metabolismo , Células Vero , Replicación ViralRESUMEN
Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.
Asunto(s)
Linfocitos B/patología , Efrinas/genética , Leucemia Linfocítica Crónica de Células B/genética , Receptores de la Familia Eph/genética , Biomarcadores de Tumor/análisis , Estudios de Casos y Controles , Efrina-A4/genética , Perfilación de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Pronóstico , Proteínas Tirosina Quinasas Receptoras/genética , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
The Eph receptor tyrosine kinases and their membrane-anchored ligands, ephrins, are signaling proteins that act as axon guidance molecules during chick auditory brainstem development. We recently showed that Eph proteins also affect patterns of neural activation in the mammalian brainstem. However, functional deficits in the brainstems of mutant mice have not been assessed physiologically. The present study characterizes neural activation in Eph protein deficient mice in the auditory brainstem response (ABR). We recorded the ABR of EphA4 and ephrin-B2 mutant mice, aged postnatal day 18-20, and compared them to wild type controls. The peripheral hearing threshold of EphA4(-/-) mice was 75% higher than that of controls. Waveform amplitudes of peak 1 (P1) were 54% lower in EphA4(-/-) mice than in controls. The peripheral hearing thresholds in ephrin-B2(lacZ/)(+) mice were also elevated, with a mean value 20% higher than that of controls. These ephrin-B2(lacZ/)(+) mice showed a 38% smaller P1 amplitude. Significant differences in latency to waveform peaks were also observed. These elevated thresholds and reduced peak amplitudes provide evidence for hearing deficits in both of these mutant mouse lines, and further emphasize an important role for Eph family proteins in the formation of functional auditory circuitry.
Asunto(s)
Vías Auditivas/metabolismo , Umbral Auditivo , Efrina-A4/metabolismo , Efrina-B2/metabolismo , Potenciales Evocados Auditivos del Tronco Encefálico , Trastornos de la Audición/metabolismo , Estimulación Acústica , Animales , Vías Auditivas/fisiopatología , Efrina-A4/deficiencia , Efrina-A4/genética , Efrina-B2/deficiencia , Efrina-B2/genética , Genotipo , Trastornos de la Audición/genética , Trastornos de la Audición/fisiopatología , Ratones , Ratones Noqueados , Fenotipo , Tiempo de Reacción , Factores de TiempoRESUMEN
BACKGROUND: ADAM15 is a metalloprotease-disintegrin implicated in ectodomain shedding and cell adhesion. Aberrant ADAM15 expression has been associated with human cancer and other disorders. We have previously shown that the alternative splicing of ADAM15 transcripts is mis-regulated in cancer cells. To gain a better understanding of ADAM15 regulation, its genomic organization and regulatory elements as well as the alternative exon use in human tissues were characterized. RESULTS: Human ADAM15, flanked by the FLJ32785/DCST1 and ephrin-A4 genes, spans 11.4 kb from the translation initiation codon to the polyadenylation signal, being the shortest multiple-exon ADAM gene. The gene contains 23 exons varying from 63 to 316 bp and 22 introns from 79 to 1283 bp. The gene appeared to have several transcription start sites and their location suggested the promoter location within a CpG island proximal to the translation start. Reporter expression experiments confirmed the location of functional GC-rich, TATAless and CAATless promoter, with the most critical transcription-supporting elements located -266 to -23 bp relative to the translation start. Normal human tissues showed different complex patterns of at least 13 different ADAM15 splice variants arising from the alternative use of the cytosolic-encoding exons 19, 20a/b, and 21a/b. The deduced ADAM15 protein isoforms have different combinations of cytosolic regulatory protein interaction motifs. CONCLUSION: Characterization of human ADAM15 gene and identification of elements involved in the regulation of transcription and alternative splicing provide important clues for elucidation of physiological and pathological roles of ADAM15. The present results also show that the alternative exon use is a physiological post-transcriptional mechanism regulating ADAM15 expression in human tissues.
Asunto(s)
Proteínas ADAM/genética , Empalme Alternativo/fisiología , Exones/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Proteínas ADAM/biosíntesis , Secuencias de Aminoácidos/genética , Adhesión Celular/fisiología , Línea Celular , Codón Iniciador/fisiología , Islas de CpG/fisiología , Efrina-A4/genética , Efrina-A4/metabolismo , Humanos , Proteínas de la Membrana/biosíntesis , Neoplasias/enzimología , Neoplasias/genética , Especificidad de Órganos/fisiología , Regiones Promotoras Genéticas/fisiología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genéticaRESUMEN
Boundaries between cellular compartments often serve as signaling interfaces during embryogenesis. The coronal suture is a major growth center of the skull vault and develops at a boundary between cells derived from neural crest and mesodermal origin, forming the frontal and parietal bones, respectively. Premature fusion of these bones, termed coronal synostosis, is a common human developmental anomaly. Known causes of coronal synostosis include haploinsufficiency of TWIST1 and a gain of function mutation in MSX2. In Twist1(+/-) mice with coronal synostosis, we found that the frontal-parietal boundary is defective. Specifically, neural crest cells invade the undifferentiated mesoderm of the Twist1(+/-) mutant coronal suture. This boundary defect is accompanied by an expansion in Msx2 expression and reduction in ephrin-A4 distribution. Reduced dosage of Msx2 in the Twist1 mutant background restores the expression of ephrin-A4, rescues the suture boundary and inhibits craniosynostosis. Underlining the importance of ephrin-A4, we identified heterozygous mutations in the human orthologue, EFNA4, in three of 81 patients with non-syndromic coronal synostosis. This provides genetic evidence that Twist1, Msx2 and Efna4 function together in boundary formation and the pathogenesis of coronal synostosis.
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Suturas Craneales/metabolismo , Craneosinostosis/metabolismo , Craneosinostosis/patología , Efrinas/metabolismo , Mesodermo/metabolismo , Cresta Neural/metabolismo , Receptores de la Familia Eph/metabolismo , Animales , Secuencia de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Efrina-A2/genética , Efrina-A2/metabolismo , Efrina-A4/genética , Efrina-A4/metabolismo , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Cresta Neural/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Transducción de Señal , Células Tumorales Cultivadas , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes an axon guiding TGF-beta, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. We present a model for the integrated control of TGF-beta, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/metabolismo , Efrina-A4/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Órganos de los Sentidos/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Distribución de Chi-Cuadrado , Mapeo Cromosómico/métodos , Clonación Molecular , Análisis Mutacional de ADN , Elementos de Facilitación Genéticos , Efrina-A4/genética , Efrinas/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Inmunohistoquímica/métodos , Proteínas Luminiscentes/metabolismo , Masculino , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Insercional/métodos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Secuencia de ADN , Factor de Crecimiento Transformador beta/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Eph receptor tyrosine kinases and ephrins are required for axon patterning and plasticity in the developing nervous system. Typically, Eph-ephrin interactions promote inhibitory events; for example, prohibiting the entry of neural cells into certain embryonic territories. Here, we show that distinct subsets of motor neurons that express EphA4 respond differently to ephrin-A5. EphA4-positive LMC(l) axons avoid entering ephrin-A5-positive hindlimb mesoderm. In contrast, EphA4-positive MMC(m) axons extend through ephrin-A5-positive rostral half-sclerotome. Blocking EphA4 activation in MMC(m) neurons or expanding the domain of ephrin-A5 expression in the somite results in the aberrant growth of MMC(m) axons into the caudal half-sclerotome. Moreover, premature expression of EphA4 in MMC(m) neurons leads to a portion of their axons growing into novel ephrin-A5-positive territories. Together, these results indicate that EphA4-ephrin-A5 signaling acts in a positive manner to constrain MMC(m) axons to the rostral half-sclerotome. Furthermore, we show that Eph activation localizes to distinct subcellular compartments of LMC(l) and MMC(m) neurons, consistent with distinct EphA4 signaling cascades in these neuronal subpopulations.
Asunto(s)
Efrina-A4/biosíntesis , Efrina-A5/fisiología , Neuronas Motoras/metabolismo , Inhibición Neural/fisiología , Animales , Axones/efectos de los fármacos , Axones/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Electroporación , Efrina-A4/genética , Efrina-A5/genética , Efrina-A5/farmacología , Miembro Posterior/embriología , Miembro Posterior/inervación , Ligandos , Mesodermo/metabolismo , Neuronas Motoras/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1, and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized fibrinogen via alpha(IIb)beta(3). Adhesion occurs more slowly than with adenosine diphosphate (ADP) and requires phosphatidylinositol 3 (PI3)-kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca(++) ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca(++); (2) this is accomplished in part by activating Rap1; and (3) these effects require oligomerization of ephrinB1 but not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain.
Asunto(s)
Plaquetas/metabolismo , Efrina-B1/metabolismo , Agregación Plaquetaria/fisiología , Transducción de Señal/fisiología , Proteínas de Unión al GTP rap1/metabolismo , Efrina-A4/genética , Efrina-A4/metabolismo , Humanos , Fosforilación , Adhesividad Plaquetaria/fisiología , Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Eph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+ progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a beta1 integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking.
Asunto(s)
Células Dendríticas/enzimología , Efrina-A2/fisiología , Fibronectinas/química , Receptores de la Familia Eph/fisiología , Antígenos CD34/análisis , Adhesión Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Movimiento Celular , Células Dendríticas/citología , Efrina-A2/biosíntesis , Efrina-A2/genética , Efrina-A4/biosíntesis , Efrina-A4/genética , Efrina-B1/biosíntesis , Efrina-B1/genética , Efrina-B3/biosíntesis , Efrina-B3/genética , Células Epidérmicas , Epidermis/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Integrina beta1/fisiología , Queratinocitos/enzimología , Células de Langerhans/citología , Células de Langerhans/enzimología , Polilisina/química , Proteínas Recombinantes de Fusión/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.
Asunto(s)
Corteza Cerebral/metabolismo , Efrina-A4/genética , Efrina-A5/genética , Receptor EphA4/genética , Receptor EphA5/genética , Tálamo/metabolismo , Animales , Mapeo Encefálico/métodos , Corteza Cerebral/embriología , Corteza Cerebral/enzimología , Efrina-A4/biosíntesis , Efrina-A4/fisiología , Efrina-A5/biosíntesis , Efrina-A5/fisiología , Femenino , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/embriología , Vías Nerviosas/enzimología , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Receptor EphA4/biosíntesis , Receptor EphA4/fisiología , Receptor EphA5/biosíntesis , Receptor EphA5/fisiología , Tálamo/embriología , Tálamo/enzimologíaRESUMEN
The formation of topographic neural maps relies on the coordinate assignment of neuronal cell body position and axonal trajectory. The projection of motor neurons of the lateral motor column (LMC) along the dorsoventral axis of the limb mesenchyme constitutes a simple topographic map that is organized in a binary manner. We show that LIM homeodomain proteins establish motor neuron topography by coordinating the mediolateral settling position of motor neurons within the LMC with the dorsoventral selection of axon pathways in the limb. These topographic projections are established, in part, through LIM homeodomain protein control of EphA receptors and ephrin-A ligands in motor neurons and limb mesenchymal cells.