Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells
Citation: Zhang Z-H, Lu Y-Y, Yue J (
Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells
Zhe-Hao Zhang 0
Ying-Ying Lu 0
Jianbo Yue 0
Austin John Cooney, Baylor College of Medicine, United States of America
0 Department of Physiology, University of Hong Kong , Hong Kong , China
Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca2+ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.
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The in vitro generation of neural cells from ES cells promises to
produce an almost unlimited supply of cells suitable for cell-based
replacement therapies in the nervous system [15]. The most
widely used method to trigger neural differentiation is to induce
embryoid body (EB) formation followed by retinoic acid (RA)
treatment [5,6], or, to culture ES cells with stroma conditioned
medium [7,8]. Several studies have been able to direct ES cell
differentiation and to generate specific neuronal populations,
including spinal cord motor neurons, dorsal interneurons,
cerebellar Purkinje and granule cells, and midbrain dopaminergic
neurons [9,10]. Because ES cells are pluripotential and readily
differentiate into almost any cell type in suspension culture, the
efficiency of neural induction by these methods is low and the final
cultures are always a heterogenous mixture of various cell types
[1]. A simple and efficient way to induce ES cells into neural
precursors and subsequently generate neuronal and glia cells is to
culture ES cells in an adherent monolayer in defined medium
[1,2]. In this method, ES cells are cultured in defined serum-free
and feeder-free conditions, in the absence of bone morphogenetic
protein (BMP) and Wnts signals. In these conditions, ES cells
undergo neural commitment through an autocrine fibroblast
growth factor (FGF) signaling mechanism. This method results in a
more efficient neural differentiation. Yet, around 40% of cells still
resist neural specification and adopt nonneural fates [1,2].
Therefore, to more efficiently induce neural commitment of ES
cells, it is essential to define novel cellular and molecular events
involved in neural differentiation.
Mobilization of intracellular Ca2+ stores is involved in almost all
the aspects of cellular processes, e.g. neural differentiation [1114].
Nicotinic adenine acid dinucleotide phosphate (NAADP) is one of
the most potent endogenous Ca2+ mobilizing messengers. NAADP
is formed by a base-exchange reaction that replaces the
nicotinamidemoiety of NADP with nicotinic acid and is catalyzed
by ADP-ribosyl cyclases (ARCs). Two enzymes have so far been
shown to be capable of synthesizing NAADP from NADP in vitro,
Aplysia ARC and CD38. Endogenous NAADP levels can be
modulated by a variety of extracellular stimuli. NAADP mobilizes
Ca2+ from an acidic lysosomes-related store, which can be
subsequently amplified into global Ca2+ waves by CICR from
ER/SR via IP3 receptors (IP3Rs) or ryanodine receptors (RyRs)
[15,16]. Recently, two pore channel 2 (TPC2), a new member of
the superfamily of voltage-gated ion channels containing 12
putative transmembrane segments, has been demonstrated to be
the NAADP receptors in many cell types. TPC2 is located on
lysosomal membranes when expressed in several cell types. TPC2
overexpression promotes NAADP-induced Ca2+ release from
lysosome-related stores, whereas ablating or knocking-down
TPC2 expression blocked NAADP-induced Ca2+ release [17
24]. Yet it has been recently shown that NAADP does not directly
bind to TPC2 [2527]. In addition, TPC1, TRPML1, TRPM2, or
RyRs has been reported to be NAADP receptor in different cell
types [2833].
NAADP/Ca2+ signaling pathway regulates diverse cellular
processes, including fertilization [34,35], receptor activation in
lymphocytes [36], insulin secretion in pancreatic isl (...truncated)