RESUMEN
The paired box and homeodomain containing transcription factors Pax4 and Pax6 are known to be essential for development of the pancreatic endocrine cells. In this report we demonstrate that stable expression of Pax4 in a rat glucagon-producing cell line inhibits the endogenously expressed glucagon gene completely. Furthermore, Pax4 represses Pax6 independent transcription of the insulin promoter, suggesting that Pax4 can actively repress transcription in addition to acting by competition with the transcriptional activator Pax6.
Asunto(s)
Regulación de la Expresión Génica , Glucagón/genética , Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/fisiología , Proteínas del Ojo , Técnica del Anticuerpo Fluorescente , Glucagón/metabolismo , Proteínas de Homeodominio/genética , Hibridación in Situ , Insulina/genética , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transfección , Células Tumorales CultivadasRESUMEN
Alpha-cell specific transcription of the glucagon gene is mainly conferred by the glucagon promoter G1-element, while additional elements G2, G3, and G4 have broad islet cell specificity. Transcription of the glucagon gene has been shown to be stimulated by Pax6 through binding to the glucagon gene promoter G3-element. In this report, we show that Pax6 additionally binds the glucagon gene promoter G1-element and forms a transcriptionally active complex with another homeodomain protein, Cdx2/3. Two distinct mutations in the G1-element, that both reduce promoter activity by 85-90%, is shown to eliminate binding of either Pax6 or Cdx2/3. Additionally, Pax6 enhanced Cdx2/3 mediated activation of a glucagon reporter in heterologous cells. We discuss how Pax6 may contribute to cell-type specific transcription in the pancreatic islets by complex formation with different transcription factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glucagón/genética , Proteínas de Homeodominio/metabolismo , Regiones Promotoras Genéticas , Células 3T3 , Secuencia de Aminoácidos , Animales , Factor de Transcripción CDX2 , Cricetinae , Proteínas del Ojo , Regulación de la Expresión Génica , Genes Reporteros , Ratones , Datos de Secuencia Molecular , Mutagénesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Ratas , Proteínas Represoras , TransactivadoresRESUMEN
The somatostatin upstream enhancer (SMS-UE) is a highly complex enhancer element. The distal A-element contains overlapping Pdx1 and Pbx binding sites. However, a point mutation in the A-element that abolishes both Pdxl and Pbx binding does not impair promoter activity. In contrast, a point mutation that selectively eliminates Pdx1 binding to a proximal B-element reduces the promoter activity. The B-element completely overlaps with a Pax6 binding site, the C-element. A point mutation in the C-element demonstrates that Pax6 binding is essential for promoter activity. Interestingly, a block mutation in the A-element reduces both Pax6 binding and promoter activity. In heterologous cells, Pdx1 potentiated Pax6 mediated activation of a somatostatin reporter. We conclude that the beta/delta-cell-specific activity of the SMS-UE is achieved through simultaneous binding of Pdx1 and Pax6 to the B- and C-elements, respectively. Furthermore, the A-element appears to stabilise Pax6 binding.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Proteínas de Saccharomyces cerevisiae , Somatostatina/genética , Transactivadores/metabolismo , Células 3T3 , Animales , Fusión Artificial Génica , Secuencia de Bases , Sitios de Unión , Factor de Transcripción CDX2 , Proteínas de Unión al ADN/genética , Proteínas del Ojo , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Ratas , Proteínas Represoras , Relación Estructura-Actividad , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Insulin promoter factor-1 (IPF1) (renamed to pancreatic-duodenal homeobox factor-1, PDX1) was originally cloned and characterized as an islet beta-cell specific insulin gene transcription factor (1) and later shown to be essential for the formation of the mature pancreas (2, 3). In the adult normal pancreas PDX1 is almost exclusively expressed in the beta-cell compartment and generally absent from the alpha-cell while it is widely expressed in the pancreatic epithelium during development. Using pluripotent rat islet tumor cultures and derived insulinomas and glucagonomas we have analyzed differential expression of a large number of genes including the transcription factors PDX1, Nkx6.1, Pax6, and NeuroD. While NeuroD and Pax6 expression was detectable among all phenotypes, PDX1 was expressed in the pluripotent culture and maintained in the insulinoma, while Nkx6.1 was selectively co-induced with insulin during insulinoma formation. Both factors were not detectable in the glucagonoma. Nkx6.1 proved to have a highly beta-cell restricted expression in the adult rat. Forced expression of recombinant PDX1 in the glucagonoma resulted in efficient transcriptional activation of the endogenous insulin and IAPP genes, but did not affect glucagon gene activity. In this hybrid alpha/beta-cell phenotype the endogenous Nkx6.1 gene remained silent. We conclude that PDX1 in synergy with NeuroD specifies part of the beta-cell phenotype including transcriptional activation of insulin and IAPP genes, but that other factors such as Nkx6.1 and Pax6 are required for additional features of the fully mature beta-cell phenotype.
Asunto(s)
Islotes Pancreáticos/fisiología , Factores de Transcripción/fisiología , Animales , Humanos , Insulina/fisiología , Fenotipo , Reacción en Cadena de la Polimerasa , RatasRESUMEN
Pancreas organogenesis is a highly regulated process, in which two anlage evaginate from the primitive gut. They later fuse, and, under the influence of the surrounding mesenchyme, the mature organ develops, being mainly composed of ductal, exocrine and endocrine compartments. Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments. The three compartments are thought to be of common endodermal origin; in contrast to earlier hypotheses, which suggested that the endocrine compartment was of neuroectodermal origin. It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation. During recent years, studies of insulin-gene regulation and, in particular, the tissue-specific transcriptional control of insulin-gene activity have provided information on pancreas development in general. The present review summarizes these findings, with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas.
Asunto(s)
Proteínas de Homeodominio , Islotes Pancreáticos/citología , Páncreas/citología , Amiloide/genética , Animales , Linfocitos B/inmunología , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 2 , Humanos , Insulina/fisiología , Polipéptido Amiloide de los Islotes Pancreáticos , Ratones , Proteínas de Transporte de Monosacáridos/fisiología , Páncreas/embriología , Ratas , Transactivadores/fisiologíaRESUMEN
Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.