Novel Evidence That Extracellular Nucleotides and Nucleosides Regulate the Expression of Heme Oxygenase 1 (HO-1) in Opposite Ways in Hematopoietic Stem/Progenitor Cells (HSPCs), Which Explains Why ATP Enhances Mobilization of HSPCs, While Its Metabolite Adenosine Inhibits This Process

Background. One of the problems with the mobilization of hematopoietic stem/progenitor cells (HSPCs) in the clinic is that a significant number of patients are poor mobilizers. In order to develop more efficient mobilization strategies, we have to better understand the mobilization process at the molecular and cellular levels. We have reported that HSPCs express heme oxygenase 1 (HO-1), which is a negative regulator of the complement cascade (ComC), which in turn is required for stem cell mobilization (Stem Cell Rev. 2015, 11:110-8, Leukemia. 2017, 31:446-458). Moreover, very recently we became interested in the novel role of extracellular nucleotides (EXNs) and nucleosides in this process (Leukemia 2018, in press, doi: 10.1038/s41375-018-0122-0). The most important EXN is adenosine triphosphate (ATP), which is involved in intracellular energy transfer but if released from activated cells into the extracellular space becomes an important signaling molecule involved in purinergic signaling. Secretion of ATP from activated cells occurs in a pannexin channel-dependent manner and is additionally augmented by a positive regulatory loop after ATP binding to the P2X7 purinergic receptor. We recently found that the ATP concentration during the mobilization process increases to micromolar concentrations in the BM microenvironment. Moreover, we found that in the extracellular BM space ATP is processed by the CD39 and CD73 ectonucleotidases to ADP, AMP, and finally to adenosine, which is also an important mediator of purinergic signaling. Hypothesis. We hypothesized that EXNs and purinergic signaling via extracellular ATP and its metabolite adenosine are involved in triggering the mobilization of HSPCs and that this effect is mediated by the expression of HO-1 in HSPCs. Materials and Methods. To address this question, 2-month-old P2X7^-/-, CD39^-/-, and CD73^-/-mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1⁺c-kit⁺lineage^- (SKL) cells circulating in PB. The secretion of ATP from BM cells was inhibited by employing the pannexin 1 blocking drug probenecid or a synthetic pannexin 1 blocking peptide. In parallel, we evaluated i) the expression of HO-1 in HSPCs by employing RQ-PCR and western blot analysis and ii) the activation of the ComC by C5a ELISA. Results. Here we provide evidence that ATP, as an EXN secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates mobilization of HSPCs. Inhibition of the pannexin 1 channel by probenecid or a pannexin 1 blocking peptide inhibited this process. Furthermore, mobilization of HSPCs was augmented in a P2X7 receptor-dependent manner, evidenced by the finding that P2X7^-/- mice were poor mobilizers. Furthermore, ATP is processed to adenosine in the extracellular space by CD39 and CD73 ectonucleotidases, and to our surprise we observed that CD73-deficient mice mobilize more HSPCs than their control wild type (WT) littermates, which indicates a novel negative role for adenosine in the mobilization process. This finding was confirmed by injecting mice with adenosine during AMD3100 administration. Finally, we found that ATP and adenosine modulate the mobilization process by regulating in opposite ways HSPC expression of HO-1, which is a negative regulator of mobilization. Therefore, while ATP downregulates expression of HO-1 in HSPCs and enhances mobilization, adenosine upregulates its expression and negatively affects the egress of HSPCs from BM into PB. Conclusions. We demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs in an HO-1-dependent manner. While ATP triggers and promotes this process, adenosine has an inhibitory effect. The opposite effects of ATP and adenosine on the mobilization process can be explained by their different effects on the regulation of HO-1 expression in HSPCs. Finally, administration of ATP as a signaling molecule (together with G-CSF or AMD3100), inhibition of CD73 or HO-1 by small-molecule antagonists may provide the basis for more efficient mobilization strategies.