A Novel Underappreciated Role for the Extracellular Adenosine Triphosphate (ATP)–P2X4 Purinergic Receptor Axis in the Homing and Engraftment of HSPCs

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but when released from activated cells into the extracellular space as extracellular ATP (eATP) it becomes a crucial mediator of the purinergic signaling network. Purinergic receptors for extracellular nucleotides (EXNs), expressed on the surface of all cells in the body, are represented by the P1, P2X, and P2Y receptor families, which are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for eATP signaling and consists of seven members (P2X1–7). We found that human and murine hematopoietic stem progenitor cells (HSPCs) highly express two members of this family, the P2X4 and P2X7 receptors. We recently reported that both are involved in optimal mobilization of HSPCs by activating Nlrp3 inflammasome (Leukemia 2020 Jun;34(6):1512-1523 and Stem Cell Rev Rep. 2019 Jun;15(3):391-403). We also reported that the P2X7 receptor expressed on the surface of HSPCs facilitates the homing and engraftment of HSPCs by increasing their responsiveness to SDF-1 gradients. Interestingly, it has been proposed that both receptors heterodimerize to exert optimal activity. Hypothesis. Since, the P2X4 and P2X7 receptors show several similar biological effects in non-hematopoietic cells, we became interested in the role of the P2X4 receptor in homing and engraftment of HSPCs. Materials and Methods. To test this hypothesis, we isolated SKL cells from P2X4-KO mice and tested them for migration in response to BM chemoattractants, including the major homing factor SDF-1. Next, we tested the short- and long-term homing of mouse BM cells after exposure to the P2X4-specific inhibitor PBS12054 in normal mice by evaluating the number of donor-derived PKH67-labeled BMMNCs and CFU-GM clonogenic progenitors isolated from recipient mouse BM 24 hours after transplantation as well as the number of day-12 colony-forming units in spleen (CFU-S) and day-12 CFU-GM clonogenic progenitors. These data were confirmed in transplant studies employing P2X4-KO bone marrow cells. In parallel, we also evaluated the recovery kinetics of leukocytes and blood platelets in the PB of transplanted animals. Finally, we also perturbed P2X4 expression in transplanted mice with PBS12054 and studied the effect on homing and engraftment of normal BM cells, as described above. Results. We found that P2X4-KO mouse HSPCs have a defect in migration in response to BM chemoattractants involved in BM homing, including the major homing factor SDF-1 as well as the supportive factors S1P and eATP. Perturbation of P2X4 expression on the surface of HSPCs led to significant defective homing and engraftment of HSPCs. Moreover, inhibition of P2X4 in the recipient mouse BM microenvironment had a similar effect. Conclusions. We identified for the first time the role of eATP–P2X4 signaling in the homing and engraftment of HSPCs. To explain this result, we conclude that the eATP–P2X4 axis is, like the eATP–P2X7 axis, a potent activator of Nlrp3 inflammasomes and that defective eATP–P2X4 signaling impairs the role of purinergic signaling and the Nlrp3 inflammasome in homing and engraftment. Moreover, our results show a similar homing and engraftment phenotype for P2X4-KO mice as that seen in P2X7-KO animals, which provides functional support for the proposed dimerization of P2X7 with P2X4 receptors and the necessary presence of both receptors for optimal function. This question is currently being addressed in our laboratory by employing the fluorescence resonance energy transfer (FRET) technique. Finally, we provide additional evidence that, in addition to SDF-1 and S1P, eATP and purinergic signaling involving P2X4 and P2X7 receptors is an important and underappreciated regulator of HSPC trafficking and a potential target for molecular optimization of both processes.