Ectopic Expression of Necdin Induces Differentiation of Mouse Neuroblastoma Cells

Abstract

Necdin is expressed predominantly in postmitotic neurons, and ectopic expression of this protein strongly suppresses cell growth. Necdin has been implicated in the pathogenesis of Prader-Willi syndrome, a human neurodevelopmental disorder associated with genomic imprinting. Here we demonstrate that ectopic expression of necdin induces a neuronal phenotype in neuroblastoma cells. Necdin was undetectable in mouse neuroblastoma N1E-115 cells under undifferentiated and differentiated conditions. N1E-115 cells transfected with necdin cDNA showed morphological differentiation such as neurite outgrowth and expression of the synaptic marker proteins synaptotagmin and synaptophysin. In addition, Western blot analysis of the retinoblastoma protein (Rb) family members Rb, p130, and p107 revealed that necdin cDNA transfectants contained an increased level of p130 and a reduced level of p107, a pattern seen in differentiated G 0 cells. The transcription factors E2F1 and E2F4 physically interacted with necdin via their carboxyl-terminal transactivation domains, but only E2F1 abrogated necdin-induced growth arrest and neurite outgrowth of neuroblastoma cells. Overexpression of E2F1 in differentiated N1E-115 cells induced apoptosis, which was antagonized by co-expression of necdin. These results suggest that necdin promotes the differentiation and survival of neurons through its antagonistic interactions with E2F1.Necdin is a 325-amino acid protein encoded in a cDNA sequence that has been isolated from a subtraction library of neurally differentiated mouse embryonal carcinoma P19 cells (1). The necdin gene is expressed in most of the terminally differentiated neurons, although its expression levels vary among neuronal cell types, being the highest in the hypothalamus and brain stem (2, 3). The human necdin gene is mapped to chromosome 15q11.2-q12, a region deleted in Prader-Willi syndrome (PWS) 1 (4 -6). PWS is a neurodevelopmental disorder associated with genomic imprinting, and its major symptoms such as feeding problems, gross obesity, and hypogonadism are consistent with a hypothalamic defect. Human and mouse necdin genes are maternally imprinted and transcribed only from the paternal allele (4, 5, 7). Necdin is not expressed in the cells prepared from PWS patients whose chromosome 15q11.2-q13 region in the paternal allele is deleted. Disruption of the paternal allele of mouse necdin gene results in early postnatal lethality (8), reduction in specific hypothalamic neurons such as luteinizing hormone-releasing hormone-and oxytocin-containing neurons, and behavioral alternations, which are reminiscent of human PWS (9). Therefore, it is likely that a defect in necdin expression is responsible, at least in part, for various clinical symptoms of PWS. However, little is known about the functional roles of necdin in neuronal differentiation and development.Accumulating evidence has suggested that necdin is a growth suppressor expressed in terminally differentiated cells. Ectopic expression of necdin suppresses the proliferation of...

Necdin Downregulates Cdc2 Expression to Attenuate Neuronal Apoptosis

“…Necdin is strongly expressed in peripheral sympathetic and sensory neurons that depend on NGF for their maturation and survival (Uetsuki et al, 1996;Takazaki et al, 2002;Kuwako et al, 2005). In addition, necdin, like Rb, interacts with both E2F1 and E2F4 Kobayashi et al, 2002). These findings suggest that necdin forms complexes with these E2F proteins and attenuates apoptosis induced by E2F-responsive proapoptotic genes in neurotrophinwithdrawn neurons.…”

“…2 F). The levels of E2F1 and Cdc2 clearly increased after KCl withdrawal, whereas those of necdin and E2F4, another target of necdin (Kobayashi et al, 2002), remained unchanged. We also analyzed the levels of the cell cyclerelated proteins cyclin A, cyclin B, and PCNA and found that these proteins remained constant in the LK condition.…”

“…Necdin, like Rb, targets E2F1 and represses E2F1-dependent transcription in osteosarcoma cells Kuwako et al, 2004). Ectopic expression of necdin inhibits E2F1-induced apoptosis of differentiated neuroblastoma cells (Kobayashi et al, 2002;Kuwako et al, 2004). However, it remains unclear whether endogenous necdin inhibits E2F1-dependent transcription and apoptosis in postmitotic neurons.…”

“…Necdin targets many regulatory proteins that control neuronal life-and-death and differentiation Kobayashi et al, 2002;Tcherpakov et al, 2002;Kuwajima et al, 2004Kuwajima et al, , 2006Kuwako et al, 2004Kuwako et al, , 2005Moon et al, 2005). Thus, necdin might serve as a hub protein in the network of proteinprotein interactions to promote and stabilize neuronal terminal differentiation.…”

Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1

“…Thus, the factors that affect E2F1 functions are able to influence neuronal differentiation. In mouse neuroblastoma cells, overexpression of necdin, a factor that interacts with E2F1 and represses E2F1 activity, leads to neuronal differentiation [45] . In vivo mouse models also suggest that E2F1 is critical for central nervous system development [46] .…”