RESUMEN
One of the master regulators of placental cell fusion in mammals leading to multi-nucleated syncytiotrophoblasts is the transcription factor GCMa. Recently, we proved that the cAMP-driven protein kinase A signaling pathway is fundamental for up-regulation of GCMa transcript levels and protein stability. Here, we show that Transducer of Regulated CREB activity (TORC1), the human co-activator of cAMP response element-binding protein (CREB), but not a dominant-negative CREB mutant, significantly up-regulates the GCMa promoter. We identified potential cAMP response element (CRE)-binding sites within the GCMa promoter upstream of the transcriptional start site. Only the CRE site at -1337 interacted strongly with CREB in promoter mapping experiments. The characterization of GCMa promoter mutants and additional bZIP-type family members demonstrated that also old astrocyte specifically-induced substance (OASIS) is able to stimulate GCMa transcription. Knockdown of endogenous CREB or OASIS in BeWo cells decreased endogenous GCMa mRNA level and activity. Overexpression of TORC1 or OASIS in choriocarcinoma cells led to placental cell fusion, accompanied by placental expression of gap junction forming protein connexin-43. Further, we show that CREB expression is replaced by OASIS expression around E12.5 suggesting that a sequential order of bZIP-type family members ensures a high rate of GCMa transcription throughout placentation.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Activación Transcripcional , Trofoblastos/metabolismo , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular , Conexina 43/metabolismo , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN , Femenino , Humanos , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Placenta/embriología , Placenta/metabolismo , Placentación/genética , Embarazo , Elementos de Respuesta , Factores de Transcripción/metabolismoRESUMEN
Members of the GCM (glial cells missing) transcription factor family have been shown to act as master regulators in different cells during mammalian and fly development being responsible for processes including gliogenesis, hematopoiesis, placental formation, and development of the parathyroidea. In the central nervous system of flies, several target genes for GCM have been reported, namely repo, pointed, and tramtrack. In mammals, two GCM genes are known (GCMa and GCMb), but the knowledge of their target genes is very limited. Here, we present for the first time a global approach aimed to identify GCMa target genes. We found 66 genes up-regulated and 11 genes down-regulated in GCMa-deficient chorionic tissue of mice at embryonic day 9.5. Moreover, we verified by quantitative reverse transcription-PCR all 11 down-regulated genes. The two most strongly down-regulated genes, integrin-alpha4 and retinoblastoma (Rb1), were further analyzed by promoter studies. Additionally, we identified down-regulation of the murine syncytin A gene, which is fundamental for syncytiotrophoblast formation. Finally, we proved strong down-regulation of integrin-alpha4 and Rb1 transcript levels by in situ hybridization in murine GCMa-deficient placentae at embryonic day 9.5. Our data demonstrate for the first time that genes encoding key regulators of placental tissue formation and architecture are regulated by GCMa.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Productos del Gen env/biosíntesis , Integrina alfa4/biosíntesis , Neuropéptidos/biosíntesis , Proteínas Gestacionales/biosíntesis , Embarazo/metabolismo , Proteína de Retinoblastoma/biosíntesis , Trofoblastos/metabolismo , Animales , Línea Celular , Corion/citología , Corion/metabolismo , Proteínas de Unión al ADN , Femenino , Productos del Gen env/genética , Humanos , Integrina alfa4/genética , Ratones , Ratones Noqueados , Neuropéptidos/genética , Embarazo/genética , Proteínas Gestacionales/genética , Proteína de Retinoblastoma/genética , Factores de Transcripción , Trofoblastos/citologíaRESUMEN
The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations.