Intracellular Ca
2+ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca
2+ changes. Here we explored the role of one endogenous Ca
2+-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca
2+ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca
2+ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.Mobilization of intracellular Ca
2+ stores is involved in diverse cell functions, including fertilization, cell proliferation, and differentiation (
1–
4). At least three endogenous Ca
2+-mobilizing messengers have been identified, including inositol trisphosphate (IP
3),
3 nicotinic adenine acid dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Similar to IP
3, cADPR can mobilize calcium release in a wide variety of cell types and species, from protozoa to animals. The cADPR-mediated Ca
2+ signaling has been indicated in a variety of cellular processes (
5–
7), from abscisic acid signaling and regulation of the circadian clock in plants, to mediating long-term synaptic depression in hippocampus.Ample evidence shows that the ryanodine receptors are the main intracellular targets for cADPR (
1,
2,
8). Ryanodine receptors (RyRs) are intracellular Ca
2+ channels widely expressed in various cells and tissues, including muscles and neurons. It is the major cellular mediator of Ca
2+-induced Ca
2+ release (CICR) in cells. There are three isoforms of ryanodine receptors: RyR1, RyR2, and RyR3, all of which have been implicated in the cADPR signaling (
1,
2,
8). However, evidence regarding cADPR acting directly on the receptors is lacking (
9). It has been suggested that accessory proteins, such as calmodulin and FK506-binding protein (FKBP), may be involved instead (
10–
15).cADPR is formed from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases. Six ADP-ribosyl cyclases have been identified so far:
Aplysia ADP-ribosyl cyclase, three sea urchin homologues (
16,
17), and two mammalian homologues, CD38 and CD157 (
18). CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase. As a novel multifunctional enzyme, CD38 catalyzes the synthesis and hydrolysis of both cADPR and NAADP, two structurally and functionally distinct Ca
2+ messengers. Virtually all mammalian tissues ever examined have been shown to express CD38. CD38 knock-out mice exhibit multiple physiological defects, ranging from impaired immune responses, metabolic disturbances, to behavioral modifications (
1,
6,
18).CD38 was originally identified as a lymphocyte differentiation antigen (
18). Indeed, CD38/cADPR has been linked to cell differentiation (
5). For example, in human HL-60 cells, CD38 expression and the consequential accumulation of cADPR play a causal role in mediating granulocytic differentiation (
19). In addition, expression of CD38 in HeLa and 3T3 cells not only increased intracellular Ca
2+ concentration but also induced cell proliferation by significantly reducing the S phase duration, leading to shortened cell doubling time (
20). The ability of cADPR to increase cell proliferation has also been observed in human T cells (
21), human hemopoietic progenitors (
22), human peripheral blood mononuclear cells (
23), human mesenchymal stem cells (
24), and murine mesangial cells (
25).The PC12 cell line was derived from rat adrenal medulla and has been used extensively as a neuronal model, since it exhibits many of the functions observed in primary neuronal cultures (
26). Most importantly, PC12 cells can be induced by nerve growth factor (NGF) to differentiate into cells with extensive neurite outgrowths, resembling neuronal dendritic trees (
26,
27). In contrast to NGF, numerous growth factors and neurotransmitters can induce the proliferation of PC12 cells instead (
26). Both IP
3 receptor- and ryanodine receptor-mediated Ca
2+ stores have been shown to be present in PC12 cells (
28–
31). The type 2 ryanodine receptor is expressed in PC12 cells and activation of the NO/cGMP pathway in PC12 cells results in calcium mobilization, which is mediated by cADPR and similar to that seen in sea urchin eggs (
32). It has been demonstrated that NAADP, another Ca
2+-mobilizing messenger, is also a potent neuronal differentiation inducer in PC12 cells, while IP
3 exhibits no such role (
33,
34). Whether cADPR is involved in the proliferation and differentiation of PC12 cells is unknown.Here we show that activation of the CD38/cADPR/Ca
2+ signaling is required for the ACh-induced proliferation in PC12 cells, while inhibition of the pathway accelerates NGF-induced neuronal differentiation. Our data indicate that cADPR is important in regulating cell proliferation and neuronal differentiation in PC12 cells.
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