Abstract
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.
Highlights
In the central nervous system, control of cell cycle progression plays an essential role in the generation of the appropriate number of neurons and the formation of functional neuronal circuits
We demonstrate by gain- and loss-of-function approaches that BM88 is sufficient and necessary for accomplishment of cell cycle exit and differentiation of Neuro 2a (N2A) cells toward a neuronal phenotype, and we provide direct evidence that BM88 activates the p53-pRb signaling pathway that controls the balance between cell proliferation and cell cycle exit
Transgene BM88 Is Appropriately Targeted in the Mitochondria and ER of N2A-BM88 Cells—BM88 expression is elevated in the developing brain upon transition of precursor cells to post-mitotic neurons [24]
Summary
N2A Stable Transfection—For transfections, the full-length porcine BM88 cDNA was subcloned into the pcDNA3 vector (Invitrogen) at HindIII and XbaI sites. Detection of pRb was performed with the G3-245 monoclonal antibody, which recognizes both the hyper- and hypophosphorylated forms of the protein (Pharmingen; diluted to a final concentration of 1 g/ml), following protein electrophoresis in a 7.5% SDS-polyacrylamide gel to separate the two forms of pRb. Peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Amersham Biosciences, and blots were developed using either the diaminobenzidine (Sigma) chromogenic system or ECL (Amersham Biosciences), according to the manufacturer’s instructions. N2A cells [26] was initiated 24 h after siRNA transfection and was estimated 48 h later by measuring the length of 1) process outgrowth under phase-contrast optics and 2) III-tubulin positive processes In both cases, 1000 cells were measured from five independent experiments, and statistical analysis was performed by the paired two-sample Student’s t test.
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