RESUMEN
Although gene splicing occurs throughout the body, the phenotype of spliceosomal defects is largely limited to specific tissues. Cerebro-costo-mandibular syndrome (CCMS) is one such spliceosomal disease, which presents as congenital skeletal dysmorphism and is caused by mutations of SNRPB gene encoding Small Nuclear Ribonucleoprotein Polypeptides B/B' (SmB/B'). This study employed in vitro cell cultures to monitor osteo- and chondro-differentiation and examined the role of SmB/B' in the differentiation process. We found that low levels of SmB/B' by knockdown or mutations of SNRPB led to suppressed osteodifferentiation in Saos-2 osteoprogenitor-like cells, which was accompanied by affected splicing of Dlx5. On the other hand, low SmB/B' led to promoted chondrogenesis in HEPM mesenchymal stem cells. Consistent with other reports, osteogenesis was promoted by the Wnt/ß-catenin pathway activator and suppressed by Wnt and BMP blockers, whereas chondrogenesis was promoted by Wnt inhibitors. Suppressed osteogenic markers by SNRPB knockdown were partly rescued by Wnt/ß-catenin pathway activation. Reporter analysis revealed that suppression of SNRPB results in attenuated Wnt pathway and/or enhanced BMP pathway activities. SNRPB knockdown altered splicing of TCF7L2 which impacts Wnt/ß-catenin pathway activities. This work helps unravel the mechanism underlying CCMS whereby reduced expression of spliceosomal proteins causes skeletal phenotypes.
Asunto(s)
Discapacidad Intelectual , Micrognatismo , Costillas/anomalías , Empalmosomas , beta Catenina , beta Catenina/genética , Diferenciación Celular/genética , Empalmosomas/genética , Proteínas Nucleares snRNP/genética , Osteogénesis/genética , Vía de Señalización Wnt/genética , Células CultivadasRESUMEN
Cerebro-costo-mandibular syndrome (CCMS) is a congenital condition with skeletal and orofacial abnormalities that often results in respiratory distress in neonates. The three main phenotypes in the thorax are posterior rib gaps, abnormal costovertebral articulation and absent ribs. Although the condition can be lethal, accurate diagnosis, and subsequent management help improve the survival rate. Mutations in the causative gene SNRPB have been identified, however, the mechanism whereby the skeletal phenotypes affect respiratory function is not well-studied due to the multiple skeletal phenotypes, lack of anatomy-based studies into the condition and rarity of CCMS cases. This review aims to clarify the extent to which the three main skeletal phenotypes in the thorax contribute to respiratory distress in neonates with CCMS. Despite the posterior rib gaps being unique to this condition and visually striking on radiographic images, anatomical consideration, and meta-analyses suggested that they might not be the significant factor in causing respiratory distress in neonates. Rather, the increase in chest wall compliance due to the rib gaps and the decrease in compliance at the costovertebral complex was considered to result in an equilibrium, minimizing the impact of these abnormalities. The absence of floating ribs is likely insignificant as seen in the general population; however, a further absence of ribs or vestigial rib formation is associated with respiratory distress and increased lethality. Based on these, we propose to evaluate the number of absent or vestigial ribs as a priority indicator to develop a personalized treatment plan based on the phenotypes exhibited.
Asunto(s)
Discapacidad Intelectual , Micrognatismo , Síndrome de Dificultad Respiratoria , Costillas/anomalías , Recién Nacido , Humanos , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Micrognatismo/complicaciones , Micrognatismo/diagnóstico , Micrognatismo/genética , Síndrome de Dificultad Respiratoria/complicacionesRESUMEN
BACKGROUND: Although splicing is an integral part of the expression of many genes in our body, genetic syndromes with spliceosomal defects affect only specific tissues. To help understand the mechanism, we investigated the expression pattern of a core protein of the major spliceosome, SmB/B' (Small Nuclear Ribonucleoprotein Polypeptides B/B'), which is encoded by SNRPB. Loss-of-function mutations of SNRPB in humans cause cerebro-costo-mandibular syndrome (CCMS) characterized by rib gaps, micrognathia, cleft palate, and scoliosis. Our expression analysis focused on the affected structures as well as non-affected tissues, using chick and mouse embryos as model animals. RESULTS: Embryos at young stages (gastrula) showed ubiquitous expression of SmB/B'. However, the level and pattern of expression became tissue-specific as differentiation proceeded. The regions relating to CCMS phenotypes such as cartilages of ribs and vertebrae and palatal mesenchyme express SmB/B' in the nucleus sporadically. However, cartilages that are not affected in CCMS also showed similar expressions. Another spliceosomal gene, SNRNP200, which mutations cause retinitis pigmentosa, was also prominently expressed in cartilages in addition to the retina. CONCLUSION: The expression of SmB/B' is spatiotemporally regulated during embryogenesis despite the ubiquitous requirement of the spliceosome, however, the expression pattern is not strictly correlated with the phenotype presentation.
Asunto(s)
Discapacidad Intelectual , Empalmosomas , Humanos , Animales , Ratones , Empalmosomas/genética , Proteínas Nucleares snRNP/genética , Ribonucleoproteínas Nucleares Pequeñas , Discapacidad Intelectual/genéticaRESUMEN
Three-dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer in vitro models for metastasising conditions such as those in vessels and effusion. However, liquid culturing is often hindered by cell adhesiveness, which causes large cell clumps. We previously described a liquid culture material, LA717, which prevents nonclonal cell adhesion and subsequent clumping, thus allowing clonal growth of spheroids in an anchorage-independent manner. Here, we examined such liquid culture cancer spheroids for the acquisition of apical-basal polarity, sensitivity to an Akt inhibitor (anticancer drug MK-2206) and interaction with ECM. The spheroids present apical plasma membrane on the surface, which originated from the failure of polarisation at the single-cell stage and subsequent defects in phosphorylated ezrin accumulation at the cell boundary during the first cleavage, failing internal lumen formation. At the multicellular stage, liquid culture spheroids presented bleb-like protrusion on the surface, which was enhanced by the activation of the PI3K/Akt pathway and reduced by PI3K/Akt inhibitors. Liquid culture spheroids exhibited slow proliferation speed and low endogenous pAkt levels compared with gel-cultured spheroids and 2D-cultured cells, explaining the susceptibility to the Akt-inhibiting anticancer drug. Subcutaneous xenografting and in vitro analysis demonstrated low viability and adhesive property of liquid culture spheroids to ECM, while migratory and invasive capacities were comparable with gel-cultured spheroids. These features agree with the low efficacy of circulating tumour spheroids in the settling step of metastasis. This study demonstrates the feature of anchorage-independent spheroids and validates liquid cultures as a useful method in cancer spheroid research.
Asunto(s)
Adhesión Celular/genética , Técnicas de Cultivo de Célula , Neoplasias/genética , Esferoides Celulares/patología , Animales , Línea Celular Tumoral , Polaridad Celular/genética , Matriz Extracelular/genética , Humanos , Ratones , Metástasis de la Neoplasia , Neoplasias/patología , Proteína Oncogénica v-akt/genética , Fosfatidilinositol 3-Quinasas/genética , Transducción de Señal/genética , Esferoides Celulares/metabolismo , Trasplante HeterólogoRESUMEN
Ribs are seldom affected by developmental disorders, however, multiple defects in rib structure are observed in the spliceosomal disease cerebro-costo-mandibular syndrome (CCMS). These defects include rib gaps, found in the posterior part of the costal shaft in multiple ribs, as well as missing ribs, shortened ribs and abnormal costotransverse articulations, which result in inadequate ventilation at birth and high perinatal mortality. The genetic mechanism of CCMS is a loss-of-function mutation in SNRPB, a component of the major spliceosome, and knockdown of this gene in vitro affects the activity of the Wnt/ß-catenin and bone morphogenic protein (BMP) pathways. The aim of the present study was to investigate whether altering these pathways in vivo can recapitulate rib gaps and other rib abnormalities in the model animal. Chick embryos were implanted with beads soaked in Wnt/ß-catenin and BMP pathway modulators during somitogenesis, and incubated until the ribs were formed. Some embryos were harvested in the preceding days for analysis of the chondrogenic marker Sox9, to determine whether pathway modulation affected somite patterning or chondrogenesis. Wnt/ß-catenin inhibition manifested characteristic rib phenotypes seen in CCMS, including rib gaps (P < 0.05) and missing ribs (P < 0.05). BMP pathway activation did not cause rib gaps but yielded missing rib (P < 0.01) and shortened rib phenotypes (P < 0.05). A strong association with vertebral phenotypes was also noted with BMP4 (P < 0.001), including scoliosis (P < 0.05), a feature associated with CCMS. Reduced expression of Sox9 was detected with Wnt/ß-catenin inhibition, indicating that inhibition of chondrogenesis precipitated the rib defects in the presence of Wnt/ß-catenin inhibitors. BMP pathway activators also reduced Sox9 expression, indicating an interruption of somite patterning in the manifestation of rib defects with BMP4. The present study demonstrates that local inhibition of the Wnt/ß-catenin and activation of the BMP pathway can recapitulate rib defects, such as those observed in CCMS. The balance of Wnt/ß-catenin and BMP in the somite is vital for correct rib morphogenesis, and alteration of the activity of these two pathways in CCMS may perturb this balance during somite patterning, leading to the observed rib defects.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Discapacidad Intelectual/genética , Micrognatismo/genética , Costillas/anomalías , Factor de Transcripción SOX9/genética , Vía de Señalización Wnt/fisiología , Proteínas Nucleares snRNP/genética , Animales , Embrión de Pollo , Condrogénesis/genética , Mutación , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Naturally hypoxic conditions in amniote embryos play important roles in normal development. We previously showed that a hypoxic condition is required to produce a sufficient amount of neural crest cells (NCCs) during embryogenesis and that promoting a hypoxic response by prolyl-hydroxylase (PHD) inhibitors increases NCCs. Given that PHD inhibitors are considered as a potential treatment for anemia and ischemic diseases, we investigated the phenotypic effect of PHD inhibitors on embryonic development. METHODS: Chick embryos were administered with PHD inhibitors prior to the induction of NCCs on day 1.5. Three main events relating to hypoxia, NCCs induction, vasculogenesis and chondrogenesis, were examined. RESULTS: PHD inhibitors caused an increase of Sox10-positive NCCs in vivo. Vasculogenesis was promoted temporarily, although rapid vasculogenesis diminished the effect by day 5 in cephalic and pharyngeal regions. Studies on chondrogenesis at day 7 showed advanced development of the otic capsule, a cartilaginous structure encapsulating the inner ear. Analysis by X-ray micro-computed-tomography (µCT) revealed smaller otic capsule, suggesting premature differentiation. This in turn, deformed the developing semicircular canals within it. Other skeletal structures such as the palate and jaw were unaffected. The localized effect on the otic capsule was considered a result of the multiple effects from the hypoxic responses, increased NCCs and promoted chondrogenesis. CONCLUSION: Given the wide range of clinical applications being considered for PHD inhibitors, this study provides crucial information to caution and guide use of PHD inhibitors when treating women of childbearing age.
Asunto(s)
Oído Interno/anomalías , Ganglios Parasimpáticos/anomalías , Inhibidores de Prolil-Hidroxilasa/efectos adversos , Animales , Diferenciación Celular/efectos de los fármacos , Embrión de Pollo , Condrogénesis/efectos de los fármacos , Oído Interno/embriología , Desarrollo Embrionario/efectos de los fármacos , Ganglios Parasimpáticos/embriología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Morfogénesis/efectos de los fármacos , Cresta Neural/efectos de los fármacosRESUMEN
Cells grown in three-dimensional (3D) cultures are more likely to have native cell-cell and cell-matrix interactions than in 2D cultures that impose mechanical constraints to cells. However, most 3D cultures utilise gel matrix which, while serving as a scaffold, limits application due to its solid and opaque nature and inconsistency in cell exposure to exogenous signals. In 3D culture without gel matrix, cells tend to adhere to each other and form clumps with necrotic zone at the centre, making them unsuitable for analyses. Here we report that addition of low-molecular-weight agar named LA717 to culture media allows cells to grow as dispersed clonal spheroids in 3D. LA717 maintains cells dispersed and settled to the bottom of the medium while keeping the medium clear with little additional viscosity, making it suitable for microscopic observation. Importantly, cancer spheroids formed in LA717-containing medium show higher sensitivity to anti-cancer drugs such as Trametinib and MK-2206 that are not as effective in 2D. Because of the small and consistent size of spheroids, cell viability and drug toxicity are readily detectable in automated imaging analysis. These results demonstrate that LA717 offers a novel 3D culture system with great in vivo reflection and practicality.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células A549 , Antineoplásicos/farmacología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HCT116 , Células HeLa , Células Hep G2 , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Piridonas/farmacología , Pirimidinonas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacosRESUMEN
Hypoxia is encountered in either pathological or physiological conditions, the latter of which is seen in amniote embryos prior to the commencement of a functional blood circulation. During the hypoxic stage, a large number of neural crest cells arise from the head neural tube by epithelial-to-mesenchymal transition (EMT). As EMT-like cancer dissemination can be promoted by hypoxia, we investigated whether hypoxia contributes to embryonic EMT. Using chick embryos, we show that the hypoxic cellular response, mediated by hypoxia-inducible factor (HIF)-1α, is required to produce a sufficient number of neural crest cells. Among the genes that are involved in neural crest cell development, some genes are more sensitive to hypoxia than others, demonstrating that the effect of hypoxia is gene specific. Once blood circulation becomes fully functional, the embryonic head no longer produces neural crest cells in vivo, despite the capability to do so in a hypoxia-mimicking condition in vitro, suggesting that the oxygen supply helps to stop emigration of neural crest cells in the head. These results highlight the importance of hypoxia in normal embryonic development.
Asunto(s)
Cabeza/embriología , Cresta Neural/citología , Aminoácidos Dicarboxílicos/farmacología , Animales , Proteínas Aviares/metabolismo , Biomarcadores/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Plasticidad de la Célula/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Hiperoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Tubo Neural/efectos de los fármacos , Tubo Neural/metabolismo , Coloración y EtiquetadoRESUMEN
Hypoxic cellular response is crucial for normal development as well as in pathological conditions in order to tolerate low oxygen. The response is mediated by Hypoxia Inducible Factors (HIFs), where the α-subunit of HIF is stabilised and able to function only in low oxygen. Prolyl hydroxylases (PHDs) are oxygen dependent dioxygenase enzymes that hydroxylate HIF-α leading to HIF degradation. Thus PHDs function as an oxygen sensor for the function of HIFs. Here we describe the mRNA expression pattern of PHDs in chick embryos. Up to embryonic day 2, PHDs are weak without specific localisation, whereas from day 3 localised expression was observed in the eye, branchial arches and dermomyotome. Later in the limb development PHDs were expressed in the perichondral mesenchyme, excluded from the developing limb cartilages.
Asunto(s)
Desarrollo Embrionario/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolil Hidroxilasas/biosíntesis , Animales , Hipoxia de la Célula/genética , Embrión de Pollo/crecimiento & desarrollo , Embrión de Pollo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/metabolismo , Prolil Hidroxilasas/genética , ProteolisisRESUMEN
Cerebro-Costo-Mandibular syndrome (CCMS) is a rare autosomal dominant condition comprising branchial arch-derivative malformations with striking rib-gaps. Affected patients often have respiratory difficulties, associated with upper airway obstruction, reduced thoracic capacity, and scoliosis. We describe a series of 12 sporadic and 4 familial patients including 13 infants/children and 3 adults. Severe micrognathia and reduced numbers of ribs with gaps are consistent findings. Cleft palate, feeding difficulties, respiratory distress, tracheostomy requirement, and scoliosis are common. Additional malformations such as horseshoe kidney, hypospadias, and septal heart defect may occur. Microcephaly and significant developmental delay are present in a small minority of patients. Key radiological findings are of a narrow thorax, multiple posterior rib gaps and abnormal costo-transverse articulation. A novel finding in 2 patients is bilateral accessory ossicles arising from the hyoid bone. Recently, specific mutations in SNRPB, which encodes components of the major spliceosome, have been found to cause CCMS. These mutations cluster in an alternatively spliced regulatory exon and result in altered SNRPB expression. DNA was available from 14 patients and SNRPB mutations were identified in 12 (4 previously reported). Eleven had recurrent mutations previously described in patients with CCMS and one had a novel mutation in the alternative exon. These results confirm the specificity of SNRPB mutations in CCMS and provide further evidence for the role of spliceosomal proteins in craniofacial and thoracic development.
Asunto(s)
Anomalías Múltiples/genética , Fisura del Paladar/genética , Discapacidad Intelectual/genética , Micrognatismo/genética , Costillas/anomalías , Proteínas Nucleares snRNP/genética , Anomalías Múltiples/fisiopatología , Adolescente , Niño , Preescolar , Fisura del Paladar/complicaciones , Fisura del Paladar/fisiopatología , Exones , Femenino , Humanos , Lactante , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/fisiopatología , Masculino , Micrognatismo/complicaciones , Micrognatismo/fisiopatología , Mutación , Costillas/crecimiento & desarrollo , Costillas/fisiopatología , Escoliosis/complicaciones , Escoliosis/genética , Escoliosis/fisiopatología , Empalmosomas/genéticaRESUMEN
The 3D culture is advantageous in reflecting the in vivo condition compared to the 2D culture; however, imaging 3D-cultured cells may be a challenge due to technical restrictions. Recent development of confocal spinning disc microscope system as well as sophisticated software has enabled us to monitor dynamism of cell movement in multiple dimensions. Here we describe the method for time-lapse imaging of 3D-cultured cancer cells co-cultured with non-cancerous cells and discuss current limitations and future perspectives.
Asunto(s)
Técnicas de Cocultivo/métodos , Microscopía Confocal/métodos , Línea Celular Tumoral , Supervivencia Celular , Humanos , Factores de TiempoRESUMEN
BACKGROUND: The cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse. METHODS: GFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study. RESULTS: In contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed. CONCLUSIONS: This culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs.
RESUMEN
Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. Here, we investigate the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins, induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors.
Asunto(s)
Tipificación del Cuerpo/genética , Movimiento Celular/genética , Proteínas de Homeodominio/fisiología , Rombencéfalo/embriología , Animales , Animales Modificados Genéticamente , Embrión de Pollo , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/fisiología , Proteínas de la Membrana/genética , Ratones , Rombencéfalo/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The Wnt/ß-catenin signalling pathway shares a key component, ß-catenin, with the cadherin-based adhesion system. The signalling function of ß-catenin is conferred by a soluble cytoplasmic pool that is unstable in the absence of a Wnt signal, whilst the adhesion function is based on a cadherin-bound, stable pool at the membrane. The cadherin complex is dynamic, allowing for cell-cell rearrangements such as epithelial-mesenchymal transition (EMT), where the complex turns over through internalisation. Potential interplay between the two pools remains poorly understood, but cadherins are generally considered negative regulators of Wnt signalling because they sequester cytoplasmic ß-catenin. Here we explore how cellular changes at EMT affect the signalling capacity of ß-catenin using two models of EMT: hepatocyte growth factor (HGF) treatment of MDCK cells, and gastrulation in embryonic development. We show that EMT not only provides a pool of signalling-competent ß-catenin following internalisation of cadherin, but also significantly facilitates activation of the Wnt pathway in response to both Wnt signals and exogenous ß-catenin. We further demonstrate that availability of ß-catenin in the cytoplasm does not necessarily correlate with Wnt/ß-catenin pathway activity, since blocking endocytosis or depleting endogenous cadherin abolishes pathway activation despite the presence of ß-catenin in the cytoplasm. Lastly we present data suggesting that cadherins are required for augmented activation of the Wnt/ß-catenin pathway in vivo. This suggests that cadherins play a crucial role in ß-catenin-dependent transcription.
Asunto(s)
Cadherinas/metabolismo , Transición Epitelial-Mesenquimal , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Perros , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Endocitosis/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Células HEK293 , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
At the initial stage of carcinogenesis, transformation occurs in a single cell within an epithelial sheet. However, it remains unknown what happens at the boundary between normal and transformed cells. Using Madin-Darby canine kidney (MDCK) cells transformed with temperature-sensitive v-Src, we have examined the interface between normal and Src-transformed epithelial cells. We show that Src-transformed cells are apically extruded when surrounded by normal cells, but not when Src cells alone are cultured, suggesting that apical extrusion occurs in a cell-context-dependent manner. We also observe apical extrusion of Src-transformed cells in the enveloping layer of zebrafish gastrula embryos. When Src-transformed MDCK cells are surrounded by normal MDCK cells, myosin-II and focal adhesion kinase (FAK) are activated in Src cells, which further activate downstream mitogen-activated protein kinase (MAPK). Importantly, activation of these signalling pathways depends on the presence of surrounding normal cells and plays a crucial role in apical extrusion of Src cells. Collectively, these results indicate that interaction with surrounding normal epithelial cells influences the signalling pathways and behaviour of Src-transformed cells.
Asunto(s)
Comunicación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Transducción de Señal , Animales , Cadherinas/metabolismo , Adhesión Celular , Polaridad Celular , Perros , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Miosina Tipo II/metabolismo , Transporte de Proteínas , Pez Cebra/metabolismo , beta Catenina/metabolismoRESUMEN
The Wnt and the bone morphogenic protein (BMP) pathways are evolutionarily conserved and essentially independent signaling mechanisms, which, however, often regulate similar biological processes. Wnt and BMP signaling are functionally integrated in many biological processes, such as embryonic patterning in Drosophila and vertebrates, formation of kidney, limb, teeth and bones, maintenance of stem cells, and cancer progression. Detailed inspection of regulation in these and other tissues reveals that Wnt and BMP signaling are functionally integrated in four fundamentally different ways. The molecular mechanism evolved to mediate this integration can also be summarized in four different ways. However, a fundamental aspect of functional and mechanistic interaction between these pathways relies on tissue-specific mechanisms, which are often not conserved and cannot be extrapolated to other tissues. Integration of the two pathways contributes toward the sophisticated means necessary for creating the complexity of our bodies and the reliable and healthy function of its tissues and organs.
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Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/fisiología , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptor Cross-Talk/fisiología , Transducción de Señal/fisiología , Células Madre/citología , Proteínas Wnt/fisiología , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Modelos BiológicosRESUMEN
Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.
Asunto(s)
Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Wnt/metabolismo , Animales , Western Blotting , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Morfogenéticas Óseas/genética , Línea Celular , Pollos , Glicosilación , Humanos , Inmunoprecipitación , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Transducción de Señal , Proteínas Wnt/genéticaRESUMEN
BACKGROUND: While the body axis is largely patterned along the anterior-posterior (A-P) axis during gastrulation, the central nervous system (CNS) shows dynamic changes in the expression pattern of Hox genes during neurulation, suggesting that the CNS refines the A-P pattern continuously after neural tube formation. This study aims at clarifying the role of somites in up-regulating Hoxb4 expression to eventually establish its final pattern and how the neural tube develops a competence to respond to extrinsic signals. RESULTS: We show that somites are required for the up-regulation of Hoxb4 in the neural tube at the level of somites 1 to 5, the anterior-most domain of expression. However, each somite immediately adjacent to the neural tube is not sufficient at each level; planar signaling is additionally required particularly at the anterior-most segments of the expression domain. We also show that the dorsal side of the neural tube has a greater susceptibility to expressing Hoxb4 than the ventral region, a feature associated with dorsalization of the neural tube by BMP signals. BMP4 is additionally able to up-regulate Hoxb4 ventrally, but the effect is restricted to the axial levels at which Hoxb4 is normally expressed, and only in the presence of retinoic acid (RA) or somites, suggesting a role for BMP in rendering the neural tube competent to express Hoxb4 in response to RA or somite signals. CONCLUSION: In identifying the collaboration between somites and neural tube competence in the induction of Hoxb4, this study demonstrates interplay between A-P and dorsal-ventral (D-V) patterning systems, whereby a specific feature of D-V polarity may be a prerequisite for proper A-P patterning by Hox genes.
Asunto(s)
Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Neurulación , Transducción de Señal , Somitos/metabolismo , Animales , Embrión de Pollo , Mesodermo/metabolismo , Tubo Neural/metabolismo , Tretinoina/metabolismo , Regulación hacia ArribaRESUMEN
While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.
Asunto(s)
Coristoma/genética , Enfermedades de los Nervios Craneales , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas del Tejido Nervioso/genética , Cresta Neural/fisiología , Ganglio del Trigémino , Nervio Trigémino/embriología , Animales , Técnicas de Cultivo de Célula , Movimiento Celular , Embrión de Pollo , ADN Complementario/genética , Cabeza , Ratones , Cresta Neural/citología , Oligonucleótidos AntisentidoRESUMEN
The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.