RESUMO
Mutations in the murine Pax-3 gene lead to a range of developmental abnormalities including deficiencies in sensory and sympathetic neurones. We have investigated Pax-3 expression during neuronal differentiation and show levels of Pax-3 DNA binding decrease upon cell cycle arrest and morphological differentiation. The fall in Pax-3 DNA binding occurs within 1 h of the induction of differentiation and is mediated in part by a decrease in Pax-3 mRNA. This decrease in Pax-3 binding activity precedes any changes in cell proliferation or morphology, suggesting that the downregulation of this transcription factor may be an important prerequisite for the differentiation of neuronal progenitor cells.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição/metabolismo , Ciclo Celular/fisiologia , Divisão Celular/genética , Linhagem Celular , Replicação do DNA/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/análise , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , RNA Mensageiro/metabolismoRESUMO
Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. In this report, we have investigated the role that Pax-3 plays in neuronal cell development by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, we found that the addition of dexamethasone led to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponded with a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibited increased cell to substrate adhesion, extended long neurite processes and expressed genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression was followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation inducted in the cell lines lacking Pax-3 could be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor was downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells, and in its absence the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation.