Concentrative Uptake of Cyclic ADP-ribose Generated by BST-1+ Stroma Stimulates Proliferation of Human Hematopoietic Progenitors

Abstract

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD؉ by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38؉ feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1 ؉ stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1 ؉ stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1 ؉ human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1 ؉ feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine-and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1 ؉ feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.The self-renewal capacity of hemopoietic progenitors (HP) 1 in the bone marrow (BM) is a fundamental process in the physiology of hemopoiesis. The role of the BM stroma in providing HP with soluble factors essential to their proliferation and differentiation is well established (1, 2). However, the nature of the signals and the mechanisms by which stromal cells regulate the behavior of HP remain largely to be defined. Particularly, it is unknown whether perturbations of the balance between growth-stimulatory and -inhibitory factors may influence the expansion and self-renewal capacity of the hemopoietic reservoir.Recently, we have demonstrated that the potent intracellular calcium mobilizer cyclic ADP-ribose (cADPR) (3, 4) features properties of a novel hemopoietic growth factor (5, 6). Coinfusion of human HP with murine stromal cells transfected with the human ectocellular ADP-ribosyl cyclase CD38 improves hemopoietic stem cells engraftment into NOD/SCID mice (7). The paracrine interaction between stroma and HP has been investigated in a transwell co-culture setting where human HP were cultured over confluent monolayers of murine stromal cells transfected with human CD38: NAD ϩ efflux from stromal cells through connexin 43-formed hemichannels (8) provides CD38 with its substrate for ectocellular production of cADPR; subsequent influx of the cyclic nucleotide into HP (both committed and uncommitted progenitors) induces intracellular calcium ([Ca 2ϩ ] i ) mobilization and cell proliferation (6). In a similar co-culture setting, influx of cADPR into target 3T3 fibroblasts and dimet...

Generation of Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate by CD38 for Ca2+ Signaling in Interleukin-8-treated Lymphokine-activated Killer Cells

“…To investigate this possibility, we examined whether there is any interdependence between cADPR and NAADP production by taking advantage of transporting cADPR and NAADP to LAK cells (supplemental Fig. S1) like other cells known to be transported (35)(36)(37). The addition of exogenous cADPR to LAK cells enhanced intracellular NAADP levels, whereas the addition of NAADP failed to increase intracellular cADPR levels (Fig.…”

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.''…”

“…P18, P24, and Ap2A are present in ADPRCpositive human HeLa cells and modify the [Ca 2ϩ ] i of intact HeLa when applied extracellularly: P18 inducing a decrease, P24 and Ap2A inducing an increase of the [Ca 2ϩ ] i . Because the coculture of human hemopoietic progenitors (HP) over ADPRC-transfected stromal layers has recently been shown to stimulate or to inhibit HP proliferation, depending on the amount of cyclase activity expressed by the stroma (18), the effects of P18, P24, and Ap2A on the ''in vitro'' proliferation of human HP were investigated. Results indicate that these dinucleotides can stimulate (Ap2A) or inhibit (P18 and P24) colony growth at submicromolar concentrations and may thus be part of the network of growth-stimulatory and -inhibitory signals between ADPRC-positive hemopoietic stroma (19) and HP in the bone marrow.…”

Interaction between Human NK Cells and Bone Marrow Stromal Cells Induces NK Cell Triggering: Role of NKp30 and NKG2D Receptors

“…It is generally accepted that BMSC may favor cell proliferation and produce soluble factors involved in myeloid precursor differentiation (25)(26)(27). Thus, we addressed the question of whether IL-2-activated NK lymphocytes can affect BMSC survival, as it may occur during a viral infection (1-4).…”

“…Because NK cells reside in the bone marrow (1), it is conceivable that they may interact with stromal cells. BMSC are thought to have a role in the regulation of hemopoietic stem cell proliferation and differentiation as well in the growth of some hemopoietic malignancies (25)(26)(27). More recently, it has been reported that MSC present within BMSC are able to differentiate into mesodermic tissues or even into epithelial cells and neurons (28 -34).…”