Ectopic Expression of Necdin Induces Differentiation of Mouse Neuroblastoma Cells*

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 G0 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 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 several cell lines (10 -12). Furthermore, necdin interacts with viral oncoproteins such as SV40 large T antigen, adenovirus E1A, and the transcription factor E2F1 (11). These characteristics of necdin resemble those of the retinoblastoma gene product (Rb), a major growth suppressor protein that is mutated in many cancer cells. Rb is believed to play a pivotal role in terminal mitosis and subsequent differentiation during neurogenesis (13). These findings prompted us to examine whether necdin also promotes neuronal differentiation through its growth inhibitory property.
N1E-115 neuroblastoma cells show differentiated characteristics in response to forced expression of growth suppressors such as Rb and its family members p107 and p130 (14,15). Furthermore, p73, a p53-related growth suppressor, induces differentiation of N1E-115 cells (16). Therefore, we have attempted to examine whether ectopic expression of necdin induces a neuronal phenotype in N1E-115 neuroblastoma cells. We here demonstrate that necdin induces morphological and biochemical markers of neurons in N1E-115 neuroblastoma cells. Furthermore, we show that necdin and its binding partner E2F1 act antagonistically in the differentiation and apoptosis of neuroblastoma cells. The present gain-of-function study provides valuable information about the functional roles of necdin in the induction and maintenance of the terminally differentiated state of neurons. nation with pRc-LacZ. The total amount of plasmid DNA was adjusted equally by adding empty pRc/CMV. Cells were cultured in the medium containing 2.5% FBS for 72 h, fixed with 0.25% glutaraldehyde, stained for ␤-galactosidase activity in situ (18), and observed using a phasecontrast microscope (IX70 -22PH, Olympus). Morphologically differentiated cells were judged by the length of neurites with the length more than 2 times the cell body diameter. For Western blot analysis, N1E-115 cells were plated (4 ϫ 10 5 cells) in 90-mm dishes, cultured overnight in the growth medium, and transfected with pcDNA6/TR carrying blasticidin-resistant gene (Invitrogen) and pRc-necdin (or pRc-necdin⌬N) (4 g each). Cells were cultured in the medium containing 5 g/ml blasticidin S (Invitrogen) for 60 h, and cell lysates were prepared.
Co-immunoprecipitation Assay-The Myc tag was added to E2F1 and E2F4 by subcloning their cDNAs into a 6ϫMyc tag plasmid (a gift from Dr. M. W. McBurney, University of Ottawa, Ottawa, Canada). cDNAs encoding Myc-tagged E2F1 (amino acids 1-430) and its COOH-terminal deletion mutant E2F1⌬C (amino acids 1-337) were inserted into pRc/CMV (Invitrogen) to construct pRc-MycE2F1 and pRc-MycE2F1⌬C, respectively. cDNAs encoding Myc-tagged E2F4 (amino acids 1-417) and its COOH-terminal deletion mutant E2F4⌬C (amino acids 1-360) were also inserted into pRc/CMV to construct pRc-MycE2F4 and pRc-MycE2F4⌬C, respectively. Combinations of these expression vectors were transiently transfected into N1E-115 cells by the calcium phosphate method, and whole cell extracts were prepared 48 h after transfection. The cell extracts were incubated for 2 h at 4°C with anti-Myc antibody (9E10) or anti-necdin antibody C2 in CSK buffer (12). The complexes were precipitated with Protein A-Sepharose (Amersham Biosciences), eluted with SDS-PAGE buffer, separated by 10% SDS-PAGE, and analyzed by immunoblotting using anti-necdin antibody NC243 and anti-Myc antibody.
Reporter Assay for E2F-dependent Transactivation-N1E-115 cells were plated at a density of 1 ϫ 10 5 cells in 35-mm dishes, cultured overnight in growth medium, and transfected with the luciferase reporter plasmid containing the promoter sequence (Ϫ241 to ϩ45) of mouse DNA polymerase ␣ catalytic subunit (20) (a gift from Dr. F. Hanaoka, Osaka University, Osaka, Japan) in PGV-B (Toyo Ink, Tokyo, Japan) (0.8 g), E2F expression vector (0.4 g of pRc-E2F1 or pcDNA3-HA-E2F4), necdin expression vector (0.2, 0.4, or 0.8 g of pRc-necdin or pRc-necdin⌬N) and pRc-LacZ (0.4 g). The amount of plasmid DNA was adjusted to 4 g by adding empty pRc/CMV. Cells were cultured for 48 h and harvested to measure luciferase activity with a luminometer (Lumat LB9501, Berthold) using a reagent kit (Toyo Ink). Transfection efficiency was normalized by ␤-galactosidase activity.
Bromodeoxyuridine Incorporation Assay-N1E-115 cells were plated at 1 ϫ 10 5 cells in 35-mm dishes and cultured in growth medium overnight. Cells were transfected with pRc-necdin, pRc-necdin⌬N, pRc-E2F1, pcDNA3-HA-E2F4 (0.4 g each), and pRc-LacZ (1.2 g). Total amount of DNA was adjusted to 4 g by adding empty pRc/CMV. Cells were cultured in the medium containing 2.5% FBS for 84 h and incubated in the presence of bromodeoxyuridine (BrdUrd) for 5 h. Cells were fixed and double-stained with anti-BrdUrd antibody (Roche Diagnostics) and the anti-␤-galactosidase antibody (1:300).
Apoptosis Assay-N1E-115 cells were grown on coverslips in 35-mm dishes and transfected with combinations of pRc-LacZ, pRc-E2F1, pcDNA3.1-E2F4, p3xFLAG-necdin, and p3xFLAG-necdin⌬N (1.5 g each) using LipofectAMINE Plus reagent (Invitrogen). p3xFLAG-necdin and p3xFLAG-necdin⌬N were constructed by inserting respective cDNAs into p3xFLAG-CMV-14 expression vector (Sigma). The cells were treated with Me 2 SO 24 h after transfection, fixed, and permeabilized as above. The cells were incubated with the anti-Flag M2 monoclonal antibody (1:300) (Sigma) and the anti-E2F1 antibody (1:500) or the anti-E2F4 antibody (1:500) for 1 h at room temperature, and expressed proteins were visualized with FITC-conjugated anti-mouse IgG and rhodamine B-conjugated anti-rabbit IgG. Nuclear morphology was examined by staining with 3.3 mM Hoechst 33342 (Molecular Probes) for 15 min at room temperature and observed by fluorescence microscopy. Nuclear DNA fragmentation was analyzed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) method (21) combined with immunostaining for Flag-tagged necdin, E2F1, or E2F4. TUNEL reactivity and each expressed protein were visualized with Texas Red and FITC, respectively.  (11), did not show these characteristics (Fig. 1A). Ectopic expression of necdin increased the number of neuron-like cells to 35% of total transfected cells, whereas 10 and 14% of cells transfected with the empty vector (control) and necdin⌬N cDNA, respectively, showed morphological differentiation (Fig. 1B). We analyzed the expression of the synaptic markers synaptophysin and synaptotagmin in necdin cDNA-transfected cells by immunocytochemistry. Both synaptophysin and synaptotagmin were induced in N1E-115 cells transfected with necdin cDNA, whereas cells transfected with the empty vector (control) or necdin⌬N cDNA contained very small amounts of these proteins (Fig. 1C). By Western blot analysis, necdin was undetected in undifferentiated and differentiated N1E-115 cells, whereas the levels of synaptophysin and synaptotagmin were gradually increased by Me 2 SO treatment (Fig. 1D). Necdin cDNA transfectants selected by blasticidin resistance contained increased levels of synaptotagmin and synaptophysin (Fig. 1E), suggesting that necdin promotes synaptic differentiation. Necdin Alters the Expression of Cell Cycle-related Proteins-We analyzed the amounts of cell cycle-related proteins in Me 2 SO-treated N1E-115 cells by Western blotting (Fig. 2A). The level of the hypophosphorylated form of Rb was higher than that of hyperphosphorylated Rb at 0 and 72 h, but the levels of hyperphosphorylated Rb remained almost consistent during the course of neuronal differentiation. We also examined the levels of other Rb family members p107 and p130, which are believed to be involved in the cell cycle control during neuronal differentiation (22)(23)(24). We found that the p107 level markedly decreased, whereas the p130 level markedly increased in response to Me 2 SO treatment. We also analyzed the levels of the transcription factors E2F1 and E2F4, both of which are binding partners of the Rb family proteins. The amount of E2F1 gradually decreased, whereas that of E2F4 remained almost consistent.

Ectopic Expression of Necdin Induces Neuronal Markers in
We next examined the expression levels of these Rb family proteins in necdin cDNA transfectants (Fig. 2B). The hypophosphorylated and hyperphosphorylated Rb levels in necdin cDNA-transfected cells were similar to or slightly higher than those of control or necdin⌬N-transfected N1E-115 cells. In contrast, the p107 level was greatly reduced, whereas the p130 level increased in necdin cDNA transfectants. Expression of E2F-regulated proteins cdc2 and cyclin A1 was strikingly reduced in necdin cDNA transfectants, and the expression levels of these proteins in undifferentiated cells and cells transfected with the empty vector or necdin⌬N cDNA remained high. The E2F1 and E2F4 levels in necdin cDNA transfectants were similar to those of differentiated N1E-115 cells (data not shown). These results suggest that necdin cDNA transfectants resemble differentiated N1E-115 cells in the expression pattern of cell cycle-regulatory proteins. Necdin Directly Interacts with E2F1 and E2F4 -We have previously demonstrated that necdin binds to E2F1 by yeast two-hybrid assay (11). We examined in vitro binding ability of necdin with E2F1 and E2F4 by using their deletion mutants fused to MBP (Fig. 3A). Necdin bound to the COOH-terminal regions of both E2F1 and E2F4 (Fig. 3B). By this assay, we were able to narrow the necdin binding regions to E2F1(amino acids 383-430) and E2F4 (amino acids 360 -417), which encompass Rb binding region as schematized in Fig. 3C. We then examined the interactions of necdin with E2F1 and E2F4 in N1E-115 neuroblastoma cells by the co-immunoprecipitation assay (Fig. 4). Expression vectors encoding Myc-tagged E2F and necdin were co-transfected into N1E-115 cells. Major bands of expressed polypeptides were detected at the positions of their predicted sizes (necdin, ϳ43 kDa; necdin⌬N, ϳ35 kDa; Myc-E2F1 and Myc-E2F4, ϳ75 kDa; Myc-E2F1⌬C and Myc-E2F4⌬C; ϳ65 kDa) (Fig. 4A). In N1E-115 cells, necdin was co-immunoprecipitated with Myc-E2F1 or Myc-E2F4, but not with Myc-E2F1⌬C or Myc-E2F4⌬C (Fig. 4, B and C, upper panels). Conversely, MycE2F1 was co-immunoprecipitated with necdin, but not with necdin⌬N (Fig. 4, B and C, lower panels). These results suggest that necdin binds to the COOHterminal transactivation domains of E2F1 and E2F4 in N1E-115 cells.
E2F1 Abrogates Necdin-induced Growth Arrest of N1E-115 Cells-To examine functional interactions of necdin with E2Fs, we carried out E2F site-dependent transcription assay in N1E-115 neuroblastoma cells using a luciferase reporter carrying a promoter region of mouse DNA polymerase ␣ catalytic subunit (20) (Fig. 5A). In this assay, E2F1 and E2F4 induced 15-and 4.5-fold activation, respectively. Co-expressed necdin reduced the E2F1-dependent activity to a maximum of ϳ50% of the control value. On the other hand, necdin suppressed E2F4dependent transcription by ϳ30% at most. The E2F bindingdefective mutant necdin⌬N did not affect E2F-dependent transcription.
We next examined whether necdin inhibits cell proliferation (S phase entry) in N1E-115 cells by BrdUrd incorporation assay (Fig. 5B) transfectants, whereas those among transfectants expressing E2F1 and E2F4 were 42 and 40%, respectively, which were slightly greater than the values of control and necdin⌬N (both 34%). The necdin-induced growth suppression was abrogated by E2F1 (36%), but not by E2F4 (19%). Neither E2F1 nor E2F4 altered the population of BrdUrd-positive cells in necdin⌬N cDNA transfectants. These results suggest that E2F1 and E2F4 exert different effects on necdin-induced growth suppression of N1E-115 cells.
E2F1 Antagonizes Necdin-induced Neurite Outgrowth of N1E-115 Cells-Several lines of evidence have suggested that E2F1 and E2F4 have distinct functions in cell cycle regulation and differentiation (25). Thus, we examined whether necdininduced morphological differentiation of N1E-115 cells is modulated by E2F1 or E2F4 (Fig. 6A). E2F1-transfected cells showed no apparent morphological changes, but co-transfected E2F1 suppressed necdin-induced neurite outgrowth. On the other hand, E2F4 per se induced neurite outgrowth, and cells transfected with both necdin and E2F4 showed morphological changes similar to those transfected with necdin alone. We then quantified the modulatory effects of E2F1 and E2F4 on necdin-induced neurite outgrowth. E2F1 slightly increased the number of cells carrying extended neurites, but it antagonized the effect of necdin in a dose-dependent manner (Fig. 6B). In contrast, E2F4 alone induced neurite outgrowth, and further increased the number of cells carrying extended neurites in combination with necdin (Fig. 6C). These results indicate that E2F1 and E2F4 exert opposite effects on necdin-induced morphological differentiation of N1E-115 cells.
Necdin Suppresses E2F1-induced Death of N1E-115 Cells-We have previously reported that ectopic expression of E2F1 induces apoptosis in postmitotic neurons, in which endogenous E2F1 levels were markedly down-regulated (26). We thus examined whether necdin has modulatory effects on E2F1-induced apoptosis in differentiated N1E-115 cells. N1E-115 cells were transfected with each expression vector alone or two vectors in combination, and subsequently treated with Me 2 SO. Necdin was accumulated in the nucleus and exerted no apparent effects on the nuclear morphology (Fig. 7A). In contrast, E2F1 induced a severe shrinkage of the nucleus, in which DNA was fragmented as detected by TUNEL (Fig. 7, A and B). Co-expression of necdin prevented differentiated N1E-115 cells from E2F1-induced apoptosis. On the other hand, overexpressed E2F4, either alone or in combination with necdin, was distributed primarily in the cytoplasm and had no apparent effect on the nuclear morphology. Quantification of apoptotic and TUNEL-positive cells revealed that necdin suppressed E2F1-induced apoptosis almost completely, whereas the E2F1 binding-defective mutant necdin⌬N had little or no anti-apoptotic effect (Fig. 7, C and D). These results indicate that necdin prevents differentiated neuroblastoma cells from E2F1induced apoptosis. DISCUSSION We have recently found that primary dorsal root ganglion neurons, which express high levels of necdin, fail to differentiate properly even in the presence of nerve growth factor when endogenous necdin is down-regulated by necdin antisense oligonucleotide (27). The antisense oligonucleotide-treated cells eventually undergo caspase-3-dependent apoptosis, suggesting that necdin is required for the terminal differentiation and survival of nerve growth factor-dependent sensory neurons. Because necdin is not expressed in neural stem cells or transformed cell lines derived from pheochromocytoma and neuroblastoma, we speculated that ectopic expression of necdin induces a neuronal phenotype in these necdin-deficient cells. We initially transferred necdin cDNA into multipotent neural stem cells prepared from the neural tube and found that necdinoverexpressing stem cells failed to undergo neuronal differentiation. 2 We thus used necdin-defective N1E-115 neuroblastoma cells, which have been often used to examine the effects of exogenous genes encoding growth suppressor proteins such as 2 H. Taniura and K. Yoshikawa, unpublished observations. Rb, p130, p107, p27 KIP1 , and p73 (14 -16). Using this cell line as a gain-of-function model, we were able to demonstrate that necdin induces characteristics of differentiated neurons such as neurite outgrowth (Fig. 1, A and B), changes of expression levels of synaptic marker proteins (Fig. 1, C and E) and cell cycle regulatory proteins (Fig. 2B), and cell cycle repression (Fig. 5B). We infer that necdin requires some complementary factors expressed in the neuronal lineage-committed cells (or partially differentiated cells in a neuronal direction) but not in multipotent stem cells.
Necdin is initially characterized as a nuclear protein (1,2) and is partly associated with the nuclear matrix (28). Necdin binds to the typical nuclear proteins E2F and p53 (11,12). Recent studies have shown that necdin is distributed in the cytoplasm of differentiated neurons and skeletal muscles (19,29). Similarly, E2F1 is primarily cytoplasmic in terminally differentiated skeletal muscles, whereas E2F4 is partitioned between cytoplasmic and nuclear compartments (30). Although p53 appears in the nucleus during neuronal differentiation, it is present mainly in the cytoplasm in mature differentiated neurons (31). These observations raise the possibility that nuclear proteins involved in cell cycle regulation are sequestered in the cytoplasm after accomplishment of terminal differentiation. We infer that necdin-induced growth arrest is attributable, at least in part, to the sequestration of E2Fs from their target sites to suppress E2F-dependent transcription, and that the suppression of E2F1-responsive genes triggers the differentiation program in neuroblastoma cells.
We have confirmed the previous finding that Me 2 SO-treated N1E-115 cells express reduced p107 and increased p130 levels (15). A similar expression pattern of these Rb family proteins was observed in necdin cDNA transfectants (Fig. 2A). These are consistent with the idea that p130 is found in quiescent cells but not in growing cells, whereas p107 is inversely regulated (32). Such changes in the expression pattern of Rb family proteins are observed during the course of neuronal differentiation of P19 embryonal carcinoma cells (30,33) and neuronal progenitor cells (23) . These findings suggest that changes in the predominance of Rb family proteins from p107 to p130 are correlated with the transition from proliferative state of neuronal progenitor cells to the postmitotic state of differentiated neurons. In contrast to the striking changes in p107 and p130 levels during neuronal differentiation, only a small change in the ratio between hypophosphorylated and hyperphosphorylated Rb was observed in differentiated N1E-115 cells (Fig. 2B). We observed that differentiated N1E-115 cells and necdin cDNA transfectants contained very low levels of cdc2 and cyclin A1, both of which are up-regulated by E2F (Fig. 2, A and B), suggesting that E2F is silenced in differentiated N1E-115 cells. Thus, the phosphorylation status of Rb may exert little or no influence on the mitotic quiescence in differentiated neuroblastoma cells expressing high levels of p130 and low levels of E2F1.
The present study has shown that necdin interacts with E2F4 as well as E2F1 (11). E2F1 is thought to promote the exit from G 0 phase (34), and its overexpression induces apoptosis in quiescent cells (35) and postmitotic neurons (26,36). We found that E2F1 abrogated necdin-induced growth arrest and morphological differentiation of N1E-115 cells (Figs. 5 and 6). In addition, differentiated N1E-115 cells overexpressing E2F1 underwent apoptosis, which was antagonized by co-expression of necdin (Fig. 7). These findings suggest that necdin plays a role in the suppression of E2F1 that promotes dedifferentiation and apoptosis during neuronal differentiation. On the other hand, E2F4 failed to affect necdin-induced growth arrest of N1E-115 cells (Fig. 5B), but promoted their neuronal differentiation (Fig. 6C). It has previously been reported that E2F4 actively promotes neuronal differentiation of PC12 pheochromocytoma cells in a cell growth-independent manner (37). Thus, E2F4, unlike E2F1, may be a participant in differentiation of neuronal lineage-committed cells. We speculate that necdin and E2F4 have similar functions to promote neuronal differentiation, although the functional significance of the physical interactions between necdin and E2F4 remains to be elucidated.
Neuronal lineage-committed cell lines derived from neuroblastoma and pheochromocytoma show differentiated characteristics in response to a variety of chemical inducers. However, these cells re-enter the cell cycle upon removal of the inducers. Thus, growth arrest of differentiated neuroblastoma and pheochromocytoma cells is believed to be reversible. On the contrary, postmitotic neurons, which are generated from neural stem cells, completely lose their ability to enter the cell cycle and survive in differentiated mature state. Because necdin is expressed in postmitotic neurons generated from neural stem cells, it is likely that necdin contributes to the blockade of cell cycle reentry of postmitotic neurons. This idea is also supported by the present findings that necdin suppresses the S phase entry (Fig. 5B) and antagonizes E2F1-induced apoptosis (Fig. 7). These results suggest that necdin is a unique growth suppressor that blocks cell cycle reentry and promotes survival of postmitotic neurons. Further studies on the functions of necdin will provide insights into the mechanisms underlying terminal differentiation of postmitotic neurons.