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In neurogenesis, little is known about signal transduction pathways upstream of gene expression however, mounting evidence suggests that calcium release from internal stores plays a critical role. We have previously demonstrated that BM88 is a neuronal lineage-specific regulator of cell cycle exit and differentiation; we now report a link between BM88 and calcium signaling. Calcium imaging experiments revealed that P2Y-induced calcium mobilization is diminished in mouse neuroblastoma Neuro 2a cells stably transfected with BM88 (N2A-BM88 cells) as compared with N2A cells or N2A cells differentiated with retinoic acid. This effect is not restricted to N2A cells but is also observed in HeLa cells that are transiently transfected with BM88, indicating that cells of both neural and non-neural origin respond similarly. Further, activation of P2Y1 but not purinergic P2X receptors induces proliferation of N2A and to a lesser extent of N2A-BM88 cells. Conversely, knockdown of BM88 facilitates N2A cell proliferation both under stimulating and non-stimulating conditions. Importantly, N2A-BM88 cells are less susceptible to apoptosis triggered by C2-ceramide and exhibit reduced C2-ceramide-induced intracellular calcium release. Higher calcium uptake from mitochondria and/or lower calcium levels inside the endoplasmic reticulum may explain the reduced calcium mobilization in response to BM88. Overall, our data reveal a novel signaling mechanism by which BM88 interferes with calcium release from inositol 1,4,5-trisphosphate-sensitive stores and exerts anti-proliferative and anti-apoptotic functions.

Histone deacetylase inhibitors arrest the growth of neuroblastoma cells and induce differentiation. Identification of target genes that co-ordinate and mediate these effects is important for understanding the function of this novel class of antitumour drugs. We report here that trichostatin-A (TSA) specifically induces the transcription of Cend1, a neuronal-lineage specific regulator of cell cycle exit and differentiation, in neuroblastoma Neuro2A cells, but not in non-neuronal cells. Furthermore, we show that knockdown of Cend1 alleviates both the anti-proliferative and differentiation effect of TSA. Our findings suggest that Cend1 is an important molecular target for HDAC inhibition.

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

BM88 is a neurone-specific protein implicated in cell cycle exit and differentiation of neuronal precursors. It is widely expressed in terminally differentiated neurones but also in neuronal progenitors, albeit in lower levels. Thus BM88 expression shows a tight correlation with the progression of progenitor cells towards neuronal differentiation. Here we report the genomic organization and proximal promoter characterization of the human and mouse BM88 genes. Both promoters lie in a CpG island, are TATA-less and have multiple transcription start sites. Deletion analysis performed on the human BM88 gene revealed an 88 bp minimal promoter fragment that is preferentially active in neural cells. Importantly, this minimal promoter is sufficient to confer specific transcriptional activity in primary neurones, but not in glial cells. Within the promoter region there are four functional Sp1-binding sites. Simultaneous mutations to all four Sp1 sites results in complete loss of promoter activity. Transactivation experiments revealed that Sp1 directly activates the BM88 promoter while activation also occurs in the presence of neurogenin-1. Characterization of the promoter elements that control neurone-specific and developmental expression of BM88 should contribute to the elucidation of the transcriptional networks that regulate the transition from a proliferative neural progenitor to a post-mitotic neurone.

Previous studies have shown that the BM88 antigen, a neuron-specific molecule, promotes the differentiation of mouse neuroblastoma cells [23] (Mamalaki A., Boutou E., Hurel C., Patsavoudi E., Tzartos S. and Matsas R. (1995) The BM88 antigen, a novel neuron-specific molecule, enhances the differentiation of mouse neuroblastoma cells. J. Biol. Chem. 270, 14201-14208). In particular, stably transfected with the BM88 cDNA, Neuro 2a cells over-expressing the BM88 antigen are morphologically distinct from their non-transfected counterparts; they exhibit enhanced process outgrowth and a slower rate of division. Moreover, they respond differentially to growth factors [10] (Gomez J., Boutou E., Hurel C., Mamalaki A., Kentroti S. , Vernadakis A. and Matsas R. (1998) Overexpression of the neuron-specific molecule BM88 in mouse neuroblastoma cells: Altered responsiveness to growth factors. J. Neurosci. Res. 51, 119-128). In order to further elucidate the role of the BM88 antigen in the differentiation of developing neurons we used the in vitro system of differentiating P19 cells which closely resembles early murine development in vivo. In this study, P19 cells were driven to the neuronal pathway with retinoic acid. We examined by immunofluorescence studies the expression of the BM88 antigen in these cells and we found that it correlates well with the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) which characterizes early differentiating post-mitotic neurons. In contrast, very few of the BM88 antigen-positive/PSA-NCAM-positive cells expressed neurofilament protein, a marker of more mature neurons. Our findings, in accordance with previously reported data, strongly suggest that the BM88 antigen is involved in the early stages of differentiation of neuronal cells.

The BM88 antigen is a neuron-specific molecule widely distributed in the mammalian nervous system. It is a 22-kDa, apparently not glycosylated, integral membrane protein, which appears early during brain development and remains at high levels in the mature animal. Here, we describe the cDNA cloning of the porcine BM88 antigen and present evidence that this protein is involved in neuroblastoma cell differentiation. The deduced protein is a novel molecule consisting of 140 amino acids and bears a putative transmembrane domain at the COOH-terminal region. The mRNA of this protein is expressed only in neural tissues, where it is restricted to neurons. Stably transfected Neuro-2a cells overexpressing the BM88 antigen exhibited a significant change in morphology, reflected by enhanced process outgrowth, and a slower rate of division. Moreover, in the presence of differentiation agents, such as sucrose and retinoic acid, an accelerated differentiation of the transfected Neuro-2a cells was observed. Especially in the presence of sucrose, the consequent overexpression of the BM88 antigen in the transfected cells resulted in their enhanced morphological differentiation accompanied by the induction of neurofilament protein expression. Our results suggest that the BM88 antigen plays a role in the differentiation of neuroblastoma cells.

Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.