Abstract
Neuroblastoma cells having stem cell-like qualities are widely employed models for the study of neural stem/progenitor cell proliferation and differentiation. We find that human BE(2)C neuroblastoma cells possess a signaling cascade initiated by Ca(2+) influx via voltage-dependent calcium channels and the N-methyl-D-aspartate (NMDA) receptor and culminating in nuclear calmodulin-dependent protein kinase IV (CaMKIV)-mediated phosphorylation and activation of the transcription factors Ca(2+)/cyclic AMP-response element-binding protein (CREB) and ATF1 (activating transcription factor-1). This pathway functions to maintain BE(2)C cells in an undifferentiated, proliferative state. Parallel to this Ca(2+)-dependent pathway is a hormone-responsive program by which retinoic acid (RA) initiates the differentiation of BE(2)C cells toward a neuronal lineage. This is evidenced by RA-dependent induction of the cell cycle inhibitor p21/Cip1 (Cdk-interacting protein 1) and cell cycle arrest, induction of the neuroblastic marker doublecortin and of the neuron-specific intermediate filament protein, peripherin, and by RA-stimulated extension of neuritic processes. During neuronal differentiation there is a complex antagonistic interplay between these two major signaling pathways. RA down-regulates expression of CaMKIV and one of its upstream activators, CaMKK1 (calmodulin-dependent protein kinase kinase 1). This is accompanied by RA-induced suppression of activating phosphorylation of CREB with a time course paralleling that of CaMKIV down-regulation. RA-induced repression of the Ca(2+)/calmodulin-dependent protein kinase kinase/CaMKIV/CREB pathway appears to be involved in regulating the timing of neuronal differentiation, as shown by the effect of RNA interference of CaMKIV to markedly accelerate RA-dependent up-regulation of p21/Cip1 and doublecortin expression and RA-promoted neurite outgrowth. RA-induced repression of the CaMKIV signaling pathway may represent an early event in retinoid-dependent neuronal differentiation.
Highlights
Grant R21NS050385. □S The on-line version of this article contains supplemental Figs
The functions of intracellular Ca2ϩ have been most intensively studied in postmitotic neurons, recent evidence has raised the possibility of developmental roles of intracellular Ca2ϩ prior to or during neuronal differentiation via regulation of the proliferation and/or differentiation of neural stem cells (NSCs)2 and restricted neural progenitors (4 –10)
calmodulin-dependent protein kinase IV (CaMKIV) is capable of promoting transcriptional activation of endogenous cyclic AMP-response element-binding protein (CREB), we examined the ability of CA-CaMKIV to enhance transcription of a luciferase reporter driven by a cAMP-response element (CRE)
Summary
CaMKIV is reported to mediate more rapid and transient phosphorylation of CREB than does activation of the mitogen-activated protein kinase cascade [32]. Its expression correlates with distinct periods of cellular proliferation, differentiation, or survival [34]. Consonant with potential developmental roles, CaMKIV is found in different types of multipotent stem and progenitor cells, including immature neutrophils, embryonic stem cells, hematopoietic stem cells, and osteoclast progenitors [35,36,37,38]. Potential developmental roles of CaMKIV are largely unexplored, several recent studies have documented participation of CaMKIV in spermiogenesis and hematopoietic stem cell maintenance [37, 39, 40], and it has been observed that CaMKIV null mice exhibit reduced numbers of Purkinje neurons following cerebellar development [41]. The research described was conducted to explore the role of the CaMKIV signaling pathway in the proliferation, differentiation, and survival of multipotent neural cells. We report here that RA represses the CaMKIV cascade during its induction of neuronal differentiation, an event that may be important for temporal coordination of the transition from neural stem/progenitor cell proliferation to a neuronally differentiated state
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