Extracellular cyclic ADP‐ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

Abstract

Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.

ADP-ribosyl cyclases generate two unusual adenine homodinucleotides with cytotoxic activity on mammalian cells

“…cADPR, both exogenously added and paracrinally produced by ADPRC ϩ stroma, has been demonstrated to behave as a hemopoietic growth factor, stimulating (via Ca 2ϩ -mediated mechanisms) the proliferation of committed (CFCs) as well as of uncommitted [long-term culture-initiating cells (LTC-IC) and stem cells] HP (7,18,21,32). The facts that the dinucleotides described here are produced together with cADPR by ADPRC ϩ cells and that they affect cell Ca 2ϩ (see above) prompted us to investigate their functional effects on the proliferation of human HP ''in vitro.''…”

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

“…A 20-l aliquot was withdrawn for assay of protein (27), while the rest of the sample was deproteinized with 10% trichloroacetic acid (23). The cADPR content of the cell extracts was analyzed by two subsequent HPLC chromatographies after addition of trace amounts of radiolabeled [H 3 ]cADPR (5 ϫ 10 3 cpm) as internal standard (23).…”

“…A 20-l aliquot was withdrawn for assay of protein (27), while the rest of the sample was deproteinized with 10% trichloroacetic acid (23). The cADPR content of the cell extracts was analyzed by two subsequent HPLC chromatographies after addition of trace amounts of radiolabeled [H 3 ]cADPR (5 ϫ 10 3 cpm) as internal standard (23). Identification of the cADPR peak in the cell extracts was confirmed by: (i) co-elution with the radioactive standard: (ii) comparison of the absorbance spectrum and elution time with standard cADPR, and (iii) disappearance of the corresponding peak in the matched CD38-hydrolyzed samples (23).…”

“…The results of this study indicated that both Cx43 and CD38 play a pivotal role in the vesicle-mediated intracellular trafficking of NAD ϩ which allows substrate availability to CD38 and accordingly cADPR generation. Moreover, it became clear that availability of NAD ϩ to CD38 which, if unrestricted, could damage the cell via perturbation of calcium homeostasis and NAD (23). Fura 2-AM, EGTA-AM, protein phosphatase inhibitors, and protein kinase inhibitors were purchased from Calbiochem (Milan, Italy).…”

A multifunctional enzyme adds to its repertoire

“…It is known that Ap2A acts synergistically with cADPR by increasing the sensitivity of the ryanodine receptor to cADPR (15). cADPR is known to stimulate the proliferation of hemopoietic progenitors by means of a calcium-mediated process (16). P18 and P24 were shown to inhibit hemopoietic progenitor cell growth, whereas Ap2A increased progenitor proliferation and displayed a synergistic effect with cADPR (9).…”