The Pro-Mobilizing Effects of UDP-Glucose—P2Y14 Purinergic Receptor Signaling Are Explained By Promoting Membrane Lipid Raft Formation to Optimize Hspcs' Responsiveness to Chemoattractants—a Potential Strategy to Optimize Hematopoietic Transplantation and Improve Transplant Outcomes

Background. Mounting evidence indicates an underappreciated role of purinergic signaling for many years in the trafficking of hematopoietic stem/progenitor cells (HSPCs). Our team is interested in the role of the purinergic receptor family and its extracellular ligands belonging to the family of purines (ATP, ADP) and pyrimidines (UTP, UDP) in hematopoiesis. These signaling molecules are members of the alarmin family being released by stressed cells and are involved in the HSPCs trafficking (Leukemia 2019;33:815-825, Leukemia 2022;36:248-256). We postulate that purinergic signaling pathways may optimize the mobilization of HSPCs and become an alternative for patients who are poor HSPCs mobilizers. Recently, we focused on the extracellular alarmine UDP-glucose that, as reported (J Clin Invest 2013;123:3420-3435), promotes G-CSF-induced mobilization of HSPCs with the capacity to achieve long-term repopulation in lethally irradiated animals. Aim of the study. We become interested in explaining at the molecular level why UDP-glucose mobilizes HSPCs. We also evaluated, for the first time, the role of this alarmin in the homing and engraftment of HSPCs and assessed the biological effects of the newly synthesized 10 more potent analog of UDP-glucose that is, MRS2690. Hypothesis. We have hypothesized that the role of UDP-glucose in stem cell trafficking could be due to promoting membrane lipid raft formation and enhancing intracellular activation of Nlrp3 inflammasome, which we reported is an essential orchestrator of HSPCs migration (Leukemia 2019;33:815-825). Materials and Methods. First, we studied Transwell chemotaxis of the murine and human bone marrow (BM) HSPCs to BM chemoattractants (SDF-1 and S1P) in the presence or absence of UDP-glucose and MRS2690. Next, mice were mobilized with G-CSF and AMD3100 combined with UDP-G or MRS2690. These purinergic signaling ligands were also employed to sensitize ex vivo BM cells to be transplanted into lethally irradiated recipients. In another set of experiments, lethally irradiated recipient mice were exposed to UDP-G or MRS2690 to see the effect of these signaling molecules promoted homing and engraftment of transplanted cells. In control experiments, we employed a specific blocking molecule of P2Y14. Finally, we investigated by confocal microscopy the effect of UDP-G and MRS2690 on the formation of membrane lipid rafts and by glow assay effects on activating intracellular Nlrp3-inflammasome. We also performed metabolic studies to identify proteins involved in these phenomena, focusing on MLR formation. Results. We report for the first time that UDP-glucose and MRS2690 significantly sensitize the migration responsiveness of murine and human HSPCs to SDF-1 and S1P gradients. Moreover, they enhance the mobilization of HSPCs in response to G-CSF and AMD3100. This strategy also allowed for lowering the dose of pro-mobilizing agents. We noticed that BM cells exposed to these P2Y14-specific agonists engraft faster in lethally irradiated recipients. In turn, exposure of recipient mice to these signaling molecules facilitated homing and engraftment of transplanted cells. At the molecular level, to explain this positive effect of UDP-glucose and MRS2690 on the migration of HSPCs, we demonstrate that P2Y14 signaling promotes the incorporation of CXCR4 and S1PR₁ receptors for SDF-1 and S1P, respectively, into membrane lipid rafts. This makes both receptors more responsive to chemotactic gradients. Finally, our metabolic studies revealed that UDP-glucose enhances the pentose phosphate cycle to synthesize lipid components for membrane lipid rafts. Conclusions. For the first time, we provide evidence that physiological purinergic alarmin UDP-glucose and synthetic, more potent MRS2690 could optimize the mobilization, homing, and engraftment of HSPCs. These effects are mediated by providing lipid components for membrane lipid rafts required for optimal migration and signaling in HSPCs and efficient activation of Nlrp3 inflammasome.