RESUMEN
HES-1 is a Hairy-related basic helix-loop-helix protein with three evolutionarily conserved regions known to define its function as a transcription repressor. The basic region, helix-loop-helix domain, and WRPW motif have been characterized for their molecular function in DNA binding, dimer formation, and corepressor recruitment, respectively. In contrast, the function conferred by a fourth conserved region, the helix 3-helix 4 (H-3/4) domain, is not known. To better understand H-3/4 domain function, we expressed HES-1 variants under tetracycline-inducible control in PC12 cells. As expected, the induced expression of moderate levels of wild-type HES-1 in PC12 cells strongly inhibited nerve growth factor-induced differentiation. This repression was dependent on the H-3/4 domain. Unexpectedly, expression of HES-1 also arrested cell growth, an effect that could be reversed upon down regulation of HES-1. Concomitant with growth arrest, there was a strong reduction in bromodeoxyuridine incorporation and PCNA protein levels, although not in cyclin D1 expression. Expression of a HES-1 protein carrying the H-3/4 domain, but not the WRPW domain, still partially inhibited both proliferation and differentiation. Transcription assays in PC12 cells directly demonstrated that the H-3/4 domain can mediate DNA-binding-dependent transcription repression, even in the absence of corepressor recruitment by the WRPW motif. HES-1 expression strongly repressed transcription of the p21(cip1) promoter, a cyclin-cyclin-dependent kinase inhibitor up regulated during NGF-induced differentiation, and the H-3/4 domain is necessary for this repression. Thus, the H-3/4 domain of HES-1 contributes to transcription repression independently of WRPW function, inhibits neurite formation, and facilitates two distinct and previously uncharacterized roles for HES-1: the inhibition of cell proliferation and the direct transcriptional repression of the NGF-induced gene, p21.
Asunto(s)
Proteínas de Homeodominio/genética , Células PC12/patología , Células PC12/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular/genética , División Celular/genética , Secuencias Hélice-Asa-Hélice , Ratas , Proteínas Represoras , Factor de Transcripción HES-1 , Transcripción GenéticaRESUMEN
HES-1 is a vertebrate homologue of the Drosophila basic helix-loop-helix (bHLH) protein Hairy, a transcriptional repressor that negatively regulates neuronal differentiation. HES-1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene MASH-1, a bHLH activator homologous to the proneural Achaete-Scute genes in Drosophila. Down-regulation of HES-1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neuronal differentiation. Here we show that the differentiation of embryonic day-17 rat hippocampal neurons in culture was coincident with a decline in HES-1 expression and DNA binding. Therefore, we examined the effect of forced expression of HES-1 and MASH-1 upon nerve growth factor (NGF) -induced differentiation in TrkA transfected hippocampal neurons. Expression of HES-1 inhibited both the intrinsic and NGF-induced neurite outgrowth, whereas MASH-1 expression increased neurite outgrowth. Strikingly, the increased hippocampal differentiation observed with MASH-1 expression is completely blocked by coexpression of HES-1. Furthermore, both wild-type HES-1 and a non-DNA binding mutant of HES-1 repressed MASH-1-dependent transcription activation. These results suggest that down-regulation of HES-1 is necessary for autonomous, growth factor-induced and MASH-1-activated hippocampal differentiation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Hipocampo/citología , Proteínas de Homeodominio/metabolismo , Neuronas/citología , Factores de Transcripción/metabolismo , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Proteínas de Homeodominio/genética , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células PC12 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor trkA , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Transcripción HES-1 , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , TransfecciónRESUMEN
In this study we explored the predictability of mandibular third molar impactions. Serial panoramic radiographs from 50 non-extraction and 15 extraction patients were traced for various angular and linear measurements. A linear discriminant function analysis was performed for each stage of third molar development. The results indicate that the earliest stages of development have very little predictive value. Accordingly, a justification for the oft-practiced enucleation procedure cannot be made. Although the more the tooth is developed, the higher is the accuracy of prediction, two earlier stages where the crown is fully formed or the roots 1/3 formed possess high predictive values. Based on our data, impaction of third molars in the mandible is a predictable event both in extraction and non-extraction patients.
Asunto(s)
Tercer Molar/diagnóstico por imagen , Radiografía Panorámica , Diente Impactado/diagnóstico por imagen , Análisis Discriminante , Estudios de Evaluación como Asunto , Humanos , Modelos Lineales , Maloclusión Clase I de Angle/terapia , Mandíbula , Tercer Molar/fisiopatología , Ortodoncia Correctiva/métodos , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Extracción DentalRESUMEN
The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process are not known. Here we show that the bHLH transcriptional repressor HES-1 is a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling.