Signaling via CaMKIV in human BE(2)C neuroblastoma cells
Feliciano, David Matthew
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To probe the role of CaMKIV in cell growth, survival and differentiation in a neural cellular context, I employed for the studies described, the multipotent and neural crest derived, human BE(2)C neuroblastoma cell line. I found that human BE(2)C neuroblastoma cells possess a calcium initiated signal transduction cascade culminating in nuclear CaMKIV phosphorylation and activation of the transcription factors CREB and ATF-1 and promotes the survival of BE(2)C neuroblastoma cells in an undifferentiated proliferative state. At least one mechanism for regulation of survival appears to be through CaMKIV mediated transcriptional regulation of the anti-apoptotic effector bcl-2. My research further identified that parallel to this calcium initiated pathway is a hormone-responsive program of gene transcription in which RA promotes bcl-2 expression and also initiates the differentiation of these cells along a neuronal lineage. This is evidenced by RA-dependent morphological changes, reduction in proliferation likely due to p21/Cip1 up-regulation as well as subsequent induction of the neuroblastic marker DCX and the neuron-specific intermediate filament protein, peripherin. I uncovered a complex interplay between these two major signaling pathways during neuronal differentiation as evidenced by rapid RA-induced down-regulation of CaMKIV expression at both mRNA and protein levels, prominent down-regulation of CaMKK1, an upstream regulator of CaMKIV activity and RA-induced suppression of activating phosphorylation of the nuclear transcription factor CREB with a time course that roughly parallels that of CaMKIV down-regulation. This RA-induced suppression of the calcium/CaMKK/CaMKIV/CREB pathway appears to be involved in regulating the timing of neuronal differentiation. This is documented by CaMKIV silencing leading to acceleration of the action of RA in up-regulating the cell cycle inhibitor p21/Cip1 and the neuroblastic marker DCX. One such potential mechanism may be CaMKIV regulation of the PP2A catalytic subunit which hypothetically may control phosphorylation status of key proteins involved in differentiation. Taken together, the present research describes an intracellular calcium dependent signal transduction cascade the retinoic acid repression of which facilitates acquisition of a neuroblastic fate in BE(2)C human neuroblastoma cells.