Stroma‐generated cyclic ADP‐ribose stimulates the expansion of early human hemopoietic progenitors by a paracrine interaction

Abstract

Both mammalian ectoenzymes CD38 and BST-1, which convert NAD + into the potent intracellular calcium regulator cyclic ADP-ribose (cADPR), are expressed in the bone marrow microenvironment. Therefore, we investigated the effect of extracellular cADPR on human longterm culture-initiating cells (LTC-IC), the most immature hemopoietic progenitors (HP). A threefold expansion of LTC-IC was elicited by exposure of cord blood mononuclear cells for 24 h either to micromolar concentrations of exogenously added cADPR or to the nanomolar concentrations of cADPR that are produced by CD38-transfected murine stromal cell lines cocultured with the HP. The latter effect was due to connexin 43-mediated release of NAD + from feeder cells and to subsequent CD38-catalyzed generation of cADPR at their outer surface. The increased [Ca 2+ ] i induced in the feeder cells by autocrinally generated cADPR, however, increased production of interferon γ, a potent hemopoiesis-inhibiting cytokine. Thus, long-term culture (5 wk) of HP over undiluted CD38 + feeders decreased LTC-IC output by 90%, whereas a fivefold increase was observed when HP were cultured over a mixed CD38 +/-feeder (1:10), whose ecto-ADP-ribosyl cyclase activity was as low as that expressed by native human stroma. These results demonstrate a new paracrine role for cADPR in the regulation of human hemopoiesis.Key words: paracrine communication • NAD + • CD38 yclic ADP-ribose (cADPR) is a potent and universal intracellular calcium mobilizer (1), present and active throughout phylogenesis from protists (2) to plants (3) and vertebrates (4). In mammals, involvement of cADPR is being demonstrated in a steadily growing number of calcium-dependent cellular processes, ranging from insulin secretion (5) to muscle contraction (6) and cell proliferation (7,8). This underscores the critical role of cADPRmediated intracellular calcium changes in regulating diverse cell functions. C Both mammalian ADP-ribosyl cyclases, enzymes converting NAD + into cADPR and nicotinamide, are expressed in the hemopoietic environment: CD38 on mature hemopoietic progenitors (HP) (9, 10) and BST-1 on stromal cells (11). This prompted us to investigate the effect of cADPR on the proliferation of human HP. In a previous paper, we demonstrated that extracellular cADPR stimulates the proliferation of human committed HP, the colony-forming cells (CFC), and that this effect is causally related to an increase of the intracellular calcium concentration ([Ca 2+ ] i ) induced by this cyclic nucleotide in the cells (12). Moreover, the increase in colony output through several generations following a brief (24 h) exposure of the HP to cADPR suggested that cADPR target cells could include also HP more immature than CFC.The aim of the present work was to study the effect of cADPR on the most immature HP, i.e., the long-term culture-initiating cells (LTC-IC). This population, which does not overlap with the more mature CFC, includes the HP capable of repopulating the irradiated host (13). Their identification, in the absenc...

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

“…Cord blood-derived mononuclear cells (CB MNC) were isolated by density gradient centrifugation, washed, and resuspended in complete medium (21). Cells (10 6 per ml) were incubated for 24 h in the presence of the various dinucleotides at concentrations ranging from 0.4 to 20 M: thereafter, aliquots of cells (1-2 ϫ 10 4 ) were seeded in hemopoietic growth factor-supplemented semisolid medium (MethoCult, Stem Cell Technologies, Vancouver).…”

“…Cells (10 6 per ml) were incubated for 24 h in the presence of the various dinucleotides at concentrations ranging from 0.4 to 20 M: thereafter, aliquots of cells (1-2 ϫ 10 4 ) were seeded in hemopoietic growth factor-supplemented semisolid medium (MethoCult, Stem Cell Technologies, Vancouver). After 2 weeks, colonies were microscopically identified according to standard criteria (CFU-E, CFU-GM, and CFU-GEM) and counted: each colony identifies a colony-forming cell (CFC) (21).…”

“…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.''…”

“…Production of growth-inhibiting P18 and P24 by CD38 ϩ 3T3, along with growth-stimulating cADPR, may be in part responsible for the inhibition of CFC and LTC-IC output observed after long-term coculture (2-5 weeks) of human HP over this feeder (21). To test this hypothesis, we took advantage of the fact that NPP and CIP digestion substantially reduce P18 and P24 toxicity on HP (40 and 30 times, respectively; Fig.…”

Evidence for a Causal Role of CD38 Expression in Granulocytic Differentiation of Human HL-60 Cells

“…3). The physiological functions that involve CD38 are equally widespread and include, for example, cell proliferation (13) and expansion of human hemopoietic progenitors (14,15). CD38 knockout mice exhibit defects in neutrophil chemotaxis (16), insulin secretion (reviewed in Ref.…”