RESUMEN
To establish functional circuitry, neurons settle down in a particular spatial domain by spacing their cell bodies, which requires proper positioning of the soma and establishing of a zone with unique connections. Deficits in this process are implicated in neurodevelopmental diseases. In this study, we examined the function of EphB6 in the development of cerebral cortex. Overexpression of EphB6 via in utero electroporation results in clumping of cortical neurons, while reducing its expression has no effect. In addition, overexpression of EphrinB2, a ligand of EphB6, also induces soma clumping in the cortex. Unexpectedly, the soma clumping phenotypes disappear when both of them are overexpressed in cortical neurons. The mutual inhibitory effect of EphB6/ EphrinB2 on preventing soma clumping is likely to be achieved via interaction of their specific domains. Thus, our results reveal a combinational role of EphrinB2/EphB6 overexpression in controlling soma spacing in cortical development.
Asunto(s)
Efrina-B2 , Receptor EphB6 , Receptor EphB6/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Cuerpo Celular/metabolismo , Neuronas/metabolismoRESUMEN
The Eph receptor tyrosine kinase member EphB6 is a pseudokinase, and similar to other pseudoenzymes has not attracted an equivalent amount of interest as its enzymatically-active counterparts. However, a greater appreciation for the role pseudoenzymes perform in expanding the repertoire of signals generated by signal transduction systems has fostered more interest in the field. EphB6 acts as a molecular switch that is capable of modulating the signal transduction output of Eph receptor clusters. Although the biological effects of EphB6 activity are well defined, the molecular mechanisms of EphB6 function remain enigmatic. In this review, we use a comparative approach to postulate how EphB6 acts as a scaffold to recruit adaptor proteins to an Eph receptor cluster and how this function is regulated. We suggest that the evolutionary repurposing of EphB6 into a kinase-independent molecular switch in mammals has involved repurposing the kinase activation loop into an SH3 domain-binding site. In addition, we suggest that EphB6 employs the same SAM domain linker and juxtamembrane domain allosteric regulatory mechanisms that are used in kinase-positive Eph receptors to regulate its scaffold function. As a result, although kinase-dead, EphB6 remains a strategically active component of Eph receptor signaling.
Asunto(s)
Receptor EphB6/metabolismo , Transducción de Señal , Regulación Alostérica , Animales , Secuencia Conservada , Humanos , Receptor EphB6/química , Receptor EphB6/genética , Dominios Homologos srcRESUMEN
The Drosophila TTK protein is involved in the processes of cell differentiation and is represented by two isoforms, TTK69 and TTK88, which have a common N-terminal BTB domain and different C-terminal sequences. Earlier, it was shown that TTK69 represses the activity of enhancers and promoters by recruiting a conserved among higher eukaryotes NURD complex to chromatin. The Mep-1 protein was found in the NURD-complex of Drosophila, and this protein can interact with the C-terminal region of TTK69. In the present study, using the yeast two-hybrid system, we mapped the interacting regions of the TTK and Mep-1 proteins. We identified regions in the unique C-terminal regions of TTK isoforms that can interact simultaneously with two regions of the Mep-1 protein. The results show that, despite the low homology of the C-terminal regions, the TTK isoform retains the ability to interact with two conserved regions of the Mep-1 protein, which suggests the functional significance of this interaction.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptor EphB6/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas , Homología de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Eph receptors are the largest group amongst the receptor tyrosine kinases and are divided into two subgroups, A and B, based on ligand binding specificities and sequence conservation. Through ligand-induced and ligand-independent activities, Ephs play central roles in diverse biological processes, including embryo development, regulation of neuronal signaling, immune responses, vasculogenesis, as well as tumor initiation, progression, and metastasis. The Eph extracellular regions (ECDs) are constituted of multiple domains, and previous structural studies of the A class receptors revealed how they interact with ephrin ligands and simultaneously mediate Eph-Eph clustering necessary for biological activity. Specifically, EphA structures highlighted a model, where clustering of ligand-bound receptors relies on two distinct receptor/receptor interfaces. Interestingly, most unliganded A class receptors also form an additional, third interface, between the ligand binding domain (LBD) and the fibronectin III domain (FN3) of neighboring molecules. Structures of B-class Eph ECDs, on the other hand, have never been reported. To further our understanding of Eph receptor function, we crystallized the EphB6-ECD and determined its three-dimensional structure using X-ray crystallography. EphB6 has important functions in both normal physiology and human malignancies and is especially interesting because this atypical receptor innately lacks kinase activity and our understanding of the mechanism of action is still incomplete. Our structural data reveals the overall EphB6-ECD architecture and shows EphB6-LBD/FN3 interactions similar to those observed for the unliganded A class receptors, suggesting that these unusual interactions are of general importance to the Eph group. We also observe unique structural features, which likely reflect the atypical signaling properties of EphB6, namely the need of co-receptor(s) for this kinase-inactive Eph. These findings provide new valuable information on the structural organization and mechanism of action of the B-class Ephs, and specifically EphB6, which in the future will assist in identifying clinically relevant targets for cancer therapy.
Asunto(s)
Receptor EphB6/ultraestructura , Receptores de la Familia Eph/ultraestructura , Línea Celular , Cristalografía por Rayos X/métodos , Efrinas/metabolismo , Fibronectinas/metabolismo , Humanos , Ligandos , Fosforilación , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Receptor EphA1/metabolismo , Receptor EphA1/ultraestructura , Receptor EphB6/metabolismo , Receptores de la Familia Eph/metabolismo , Transducción de SeñalRESUMEN
Adolescent alcohol drinking is widely recognized as a significant public health problem, and evidence is accumulating that sufficient levels of consumption during this critical period of brain development have an enduring impact on neural and behavioral function. Recent studies have indicated that adolescent intermittent ethanol (AIE) exposure alters astrocyte function, astrocyte-neuronal interactions, and related synaptic regulation and activity. However, few of those studies have included female animals, and a broader assessment of AIE effects on the proteins mediating astrocyte-mediated glutamate dynamics and synaptic function is needed. We measured synaptic membrane expression of several such proteins in the dorsal and ventral regions of the hippocampal formation (DH, VH) from male and female rats exposed to AIE or adolescent intermittent water. In the DH, AIE caused elevated expression of glutamate transporter 1 (GLT-1) in both males and females, elevated postsynaptic density 95 expression in females only, and diminished NMDA receptor subunit 2A expression in males only. AIE and sex interactively altered ephrin receptor A4 (EphA4) expression in the DH. In the VH, AIE elevated expression of the cystine/glutamate antiporter and the glutamate aspartate transporter 1 (GLAST) in males only. Compared to males, female animals expressed lower levels of GLT-1 in the DH and greater levels of ephrin receptor B6 (EphB6) in the VH, in the absence of AIE effects. These results support the growing literature indicating that adolescent alcohol exposure produces long-lasting effects on astrocyte function and astrocyte-neuronal interactions. The sex and subregion specificity of these effects have mechanistic implications for our understanding of AIE effects generally.
Asunto(s)
Astrocitos/metabolismo , Etanol/administración & dosificación , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Homólogo 4 de la Proteína Discs Large/metabolismo , Femenino , Homeostasis/efectos de los fármacos , Humanos , Masculino , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor EphB6/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
Vitamin K is essential for blood coagulation and plays important roles in bone and cardiovascular health. Menaquinone-7 (MK-7) is one form of vitamin K that is especially useful due to its long half-life in the circulation. MK-7 is difficult to make via organic synthesis, and is thus commonly produced by fermentation. This study aimed to genetically modify Bacillus subtilis cultures to increase their MK-7 yield and reduce production costs. We constructed 12 different strains of B. subtilis 168 by overexpressing different combinations of the rate-limiting enzymes Dxs, Dxr, Idi, and MenA. We observed an 11-fold enhancement of production in the best-performing strain, resulting in 50 mg/L MK-7. Metabolite analysis revealed new bottlenecks in the pathway at IspG and IspH, which suggest avenues for further optimization. This work highlights the usefulness of Bacillus subtilis for industrial production of high value compounds.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Receptor EphB6/metabolismo , Transducción de Señal/fisiología , Vitamina K 2/análogos & derivados , Medios de Cultivo/metabolismo , Fermentación/fisiología , Ingeniería Metabólica/métodos , Vitamina K 2/metabolismoRESUMEN
EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.
Asunto(s)
Glándulas Suprarrenales/enzimología , Células Cromafines/enzimología , Epinefrina/biosíntesis , Receptor EphB6/fisiología , Transcripción Genética/fisiología , Tirosina 3-Monooxigenasa/genética , Regulación hacia Arriba/fisiología , Glándulas Suprarrenales/citología , Animales , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Elementos de Facilitación Genéticos , Epinefrina/metabolismo , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor EphB6/genética , Transducción de Señal , Testosterona/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) is a member of the receptor tyrosine kinase family. We previously demonstrated that EPHB6 knockout reduces catecholamine secretion in male but not female mice, and castration reverses this phenotype. We showed here that male EPHB6 knockout adrenal gland chromaffin cells presented reduced acetylcholine-triggered Ca2+ influx. Such reduction depended on the non-genomic effect of testosterone. Increased large conductance calcium-activated potassium channel current densities were recorded in adrenal gland chromaffin cells from male EPHB6 knockout mice but not from castrated knockout or female knockout mice. Blocking of the large conductance calcium-activated potassium channel in adrenal gland chromaffin cells from male knockout mice corrected their reduced Ca2+ influx. We conclude that the absence of EPHB6 and the presence of testosterone would lead to augmented large conductance calcium-activated potassium channel currents, which limit voltage-gated calcium channel opening in adrenal gland chromaffin cells. Consequently, acetylcholine-triggered Ca2+ influx is reduced, leading to lower catecholamine release in adrenal gland chromaffin cells from male knockout mice. This explains the reduced resting-state blood catecholamine levels, and hence the blood pressure, in male but not female EPHB6 knock mice. These findings have certain clinical implications.
Asunto(s)
Células Cromafines/efectos de los fármacos , Epinefrina/metabolismo , Receptor EphB6/genética , Testosterona/farmacología , Acetilcolina/farmacología , Glándulas Suprarrenales/citología , Animales , Calcio/metabolismo , Catecolaminas/metabolismo , Células Cultivadas , Células Cromafines/citología , Células Cromafines/metabolismo , Femenino , Transporte Iónico/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor EphB6/deficienciaRESUMEN
While impressive improvements have been achieved in T-ALL therapy, current treatment approaches fail in approximately 25% of patients and these patients have limited treatment options. Another significant group of patients is being overtreated, which causes long-lasting side effects. Identification of molecules controlling drug resistance in T-ALL is crucial for treatment optimisation in both scenarios. We report here the EphB6 receptor is frequently overexpressed in T-ALL. Remarkably, our observations indicate that EphB6 acts in T-ALL cells to enhance sensitivity to a DNA-damaging drug, doxorubicin, as interruption of EphB6 activity interferes with the efficiency of doxorubicin-induced eradication of T-ALL cells in cell culture and in xenograft animals. This effect relies on the protection of Akt kinase signaling, while Akt inhibition combined with doxorubicin application produces synergistic effects on the elimination of EphB6-deficient T-ALL cells. These data imply that EphB6 suppresses T-ALL resistance by interfering with Akt activity. Our observations highlight a novel role for EphB6 in reducing drug resistance of T-ALL and suggest that doxorubicin treatment should produce better results if personalised based on EphB6 levels. If successfully verified in clinical studies, this approach should improve outcomes for T-ALL patients resistant to current therapies and for patients, who are being overtreated.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor EphB6/metabolismo , Animales , Antibióticos Antineoplásicos/efectos adversos , Niño , Doxorrubicina/efectos adversos , Xenoinjertos , HumanosRESUMEN
Follicular T helper (TFH) cells orchestrate the germinal center (GC) reaction locally. Local mechanisms regulating their dynamics and helper functions are not well defined. Here we found that GC-expressed ephrin B1 (EFNB1) repulsively inhibited T cell to B cell adhesion and GC TFH retention by signaling through TFH-expressed EPHB6 receptor. At the same time, EFNB1 promoted interleukin-21 production from GC TFH cells by signaling predominantly through EPHB4. Consequently, EFNB1-null GCs were associated with defective production of plasma cells despite harboring excessive TFH cells. In a competitive GC reaction, EFNB1-deficient B cells more efficiently interacted with TFH cells and produced more bone-marrow plasma cells, likely as a result of gaining more contact-dependent help. Our results reveal a contact-dependent repulsive guidance system that controls GC TFH dynamics and effector functions locally.
Asunto(s)
Efrina-B1/metabolismo , Centro Germinal/citología , Centro Germinal/inmunología , Transducción de Señal , Linfocitos T/inmunología , Animales , Adhesión Celular , Efrina-B1/deficiencia , Humanos , Interleucinas/biosíntesis , Ratones , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptor EphB4/metabolismo , Receptor EphB6/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors comprise the most abundant receptor tyrosine kinase family characterized to date in mammals including humans. These proteins are involved in axon guidance, tissue organization, vascular development and the intricate process of various diseases including cancer. These diverse functions of EPH receptors are attributed, in part, to their abilities for heterodimerization. While the interacting partners of kinase-deficient EPHB6 receptor have been characterized, the interaction of the kinase-dead EPHA10 with any other receptor has not been identified. By using co-immunoprecipitation, we demonstrated physical interaction between kinase-deficient EPHA10 with kinase-sufficient EPHA7 receptor. Immunocytochemical analyses have revealed that these two receptors co-localize on the cell surface, and soluble portions of the receptors exist as a complex in the cytoplasm as well as the nuclei. While EPHA7 and EPHA10 co-localize similarly on the membrane in MCF10A and MCF7 cells, they were differentially co-localized in MDA-MB-231 cells stably transfected with empty pcDNA vector (MDA-MB-231-PC) or an expression construct of EPHB6 (MDA-MB-231-B6). The full-length isoforms of these receptors were co-localized on the cell surface, and the soluble forms were present as a complex in the cytoplasm as well as the nucleus in MDA-MB-231-PC cells. MDA-MB-231-B6 cells, on the other hand, were distinguished by the absence of any signal in the nuclei. Our results represent the first demonstration of physical interaction between EPHA10 and EPHA7 and their cellular co-localization. Furthermore, these observations also suggest gene-regulatory functions of the complex of the soluble forms of these receptors in breast carcinoma cells of differential invasiveness.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptor EphA7/metabolismo , Receptor EphB6/genética , Receptores de la Familia Eph/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Unión Proteica , Isoformas de Proteínas , Transporte de ProteínasRESUMEN
Anoikis is a specific type of apoptosis induced by detachment of epithelial cells from extracellular matrix, and acquiring resistance to anoikis is an important step that enables cancer cells to metastasize. EphA2, which is overexpressed in a variety of human cancers, is phosphorylated by Akt on serine 897 and mediates ligand ephrin-independent promotion of anoikis resistance through the RhoG activator Ephexin4. EphB6 is frequently silenced in invasive and metastatic cancers; however, its role in cancer progression is poorly understood. Here we show that EphB6 interacts with EphA2 and suppresses EphA2-mediated promotion of anoikis resistance in MCF7 breast cancer cells. On the other hand, knockdown of EphB6 promotes anoikis resistance. We further show that expression of EphB6 decreases serine 897 phosphorylation of EphA2 and suppresses EphA2-Ephexin4 interaction and the RhoG activation. These findings implicate EphB6 as a negative regulator of EphA2 oncogenic signaling.
Asunto(s)
Anoicis , Receptor EphA2/metabolismo , Receptor EphB6/metabolismo , Transducción de Señal , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligandos , Células MCF-7 , Fosforilación , Fosfoserina/metabolismo , Estructura Terciaria de Proteína , Receptor EphA2/química , Receptor EphB6/química , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signalling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative mutant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.
Asunto(s)
Clatrina/metabolismo , Regulación hacia Abajo/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor EphB6/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Endosomas/metabolismo , Efrina-B2/metabolismo , Células HEK293 , Humanos , Ligandos , Lisosomas/metabolismo , Unión Proteica/fisiología , Receptores de la Familia Eph/metabolismoRESUMEN
The ephrin family of receptors (Eph) and their ephrin ligands are involved in pain associated hyperalgesia, but the underlying mechanisms involved have not been fully elucidated. The EphB6 receptor is a distinctive member of the EphB subclass in that its kinase domain contains several alterations in the conserved amino acids and thus lacks catalytic activity. We sought to identify a role for EphB6 in inflammatory pain, with the murine dextran sulfate sodium (DSS) colitis model of inflammatory bowel disease (IBD). Colitis, induced with the administration of 4% (wt./vol.) DSS in the drinking water, significantly decreased EphB6 protein expression levels in neurons of the lower thoracic superficial layers of spinal dorsal horns, the location of neurons that receive the majority of nociceptive information from the colon, via the primary afferents. A shift towards increased EphB/ephrinB forward signaling, mediated by EphB6 down-regulation in neurons of the dorsal horn, may play a role in inflammatory pain caused by IBD.
Asunto(s)
Colitis/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Células del Asta Posterior/metabolismo , Receptor EphB6/biosíntesis , Enfermedad Aguda , Animales , Colitis/inducido químicamente , Colitis/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/patologíaRESUMEN
Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%). Two point mutations led to instable proteins. An in frame deletion mutation (del915-917) showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/genética , Mutación Puntual , Receptor EphB6/genética , Secuencia de Bases , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cartilla de ADN , Humanos , Neoplasias Pulmonares/patología , Fenotipo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.
Asunto(s)
Presión Sanguínea/fisiología , Hipertensión/metabolismo , Músculo Liso Vascular/enzimología , Receptor EphB6/metabolismo , Testosterona/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Femenino , Humanos , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Orquiectomía , ARN Interferente Pequeño/farmacología , Receptor EphB6/genética , Transducción de Señal/fisiología , Vasoconstricción/fisiologíaRESUMEN
Pre-clinical studies provide compelling evidence that Eph family receptor tyrosine kinases (RTKs) and ligands promote cancer growth, neovascularization, invasion, and metastasis. Tumor suppressive roles have also been reported for the receptors, however, creating a potential barrier for clinical application. Determining how these observations relate to clinical outcome is a crucial step for translating the biological and mechanistic data into new molecularly targeted therapies. We investigated eph and ephrin expression in human breast cancer relative to endpoints of overall and/or recurrence-free survival in large microarray datasets. We also investigated protein expression in commercial human breast tissue microarrays (TMA) and Stage I prognostic TMAs linked to recurrence outcome data. We found significant correlations between ephA2, ephA4, ephA7, ephB4, and ephB6 and overall and/or recurrence-free survival in large microarray datasets. Protein expression in TMAs supported these trends. While observed no correlation between ephrin ligand expression and clinical outcome in microarray datasets, ephrin-A1 and EphA2 protein co-expression was significantly associated with recurrence in Stage I prognostic breast cancer TMAs. Our data suggest that several Eph family members are clinically relevant and tractable targets for intervention in human breast cancer. Moreover, profiling Eph receptor expression patterns in the context of relevant ligands and in the context of stage may be valuable in terms of diagnostics and treatment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Efrinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama/genética , Efrina-A1/genética , Efrina-A1/metabolismo , Efrinas/genética , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Receptor EphA2/genética , Receptor EphA2/metabolismo , Receptor EphA4/genética , Receptor EphA4/metabolismo , Receptor EphA7/genética , Receptor EphA7/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptor EphB6/genética , Receptor EphB6/metabolismo , Análisis de Matrices TisularesRESUMEN
Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Perfilación de la Expresión Génica , MicroARNs/genética , Receptor EphB6/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , TransfecciónRESUMEN
Eph receptor tyrosine kinases and their ligands, ephrins, are membrane proteins coordinating a wide range of biological functions both in developing embryos and in adult multicellular organisms. Numerous studies have implicated Eph receptors in the induction of opposing responses, including cell adhesion or repulsion, support or inhibition of cell proliferation and cell migration, and progression or suppression of multiple malignancies. Similar to other receptor tyrosine kinases, Eph receptors rely on their ability to catalyze tyrosine phosphorylation for signal transduction. Interestingly, however, Eph receptors also actively utilize three kinase-deficient receptor tyrosine kinases, EphB6, EphA10, and Ryk, in their signaling network. The accumulating evidence suggests that the unusual flexibility of the Eph family, allowing it to initiate antagonistic responses, might be partially explained by the influence of the kinase-dead participants and that the exact outcome of an Eph-mediated action is likely to be defined by the balance between the signaling of catalytically potent and catalytically null receptors. We discuss in this minireview the emerging functions of the kinase-dead EphB6, EphA10, and Ryk receptors both in normal biological responses and in malignancy, and analyze currently available information related to the molecular mechanisms of their action in the context of the Eph family.