The influence of "yin and yang" of purinergic receptors of the P2X and P1 type on the hematopoietic stem cell compartment

Purinergic signaling has emerged as a crucial regulatory network in hematopoiesis that affects the migration and proliferation of hematopoietic stem/progenitor cells (HSPCs). The central purinergic mediator is adenosine triphosphate (ATP), which, after releasing from the cells into the hematopoietic microenvironment as extracellular ATP (eATP), binds as a signaling ligand to the family of P2X purinergic receptors and promotes migration and proliferation of HSPCs. Subsequently, eATP is degraded in extracellular space by two cell surface-expressed ectonucleotidases CD39 and CD73 to its metabolite extracellular adenosine (eAdo), which in the negative feedback mechanism inhibits the positive effects of eATP. We found that murine and human HSPCs express at a high level three eATP binding functional P2X receptors - P2X1, P2X4, and P2X7, and at the same time, they express highly two P1 receptors for eAdo – A2A and A2B. Based on our preliminary data, we hypothesize that while eATP engaging P2X receptors stimulates intracellular pattern recognition receptor (PPR) Nlrp3 inflammasome (Nlrp3), eAdo signaling through P1 receptors has an opposite inhibitory effect on this PRR. Our novel data demonstrated that within the beneficial physiological range of activation in the so-called “hormetic zone,” Nlrp3 stimulates the trafficking and proliferation of HSPCs. It becomes activated by reactive oxygen species (ROS) and inhibited by heme oxygenase-1 (HO-1). While the intracellular level of ROS increases in cells stimulated by eATP, the intracellular level of HO-1 in cytosol increases in response to eAdo. Therefore, in this proposal, we would like to investigate in detail the positive (“yin”) P2 receptor-mediated and negative (“yang”) P1 receptor-mediated signaling effects on Nlrp3 inflammasome mediated HSPCs trafficking. We will perform our experiments using murine and human HSPCs, murine strains deficient in purinergic receptors, and employ small molecular modulators of investigated pathways. In parallel, we will analyze in-depth molecular changes at the mRNA and protein level by using state-of-the-art OMICS strategies. These studies will lead to the development of better stem cell mobilization and homing protocols and lead to more efficient ex vivo expansion of HSPCs. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, PRJNA1128409 The mass spectrometry lipidomic data sets have been uploaded to the MassIVE repository with the dataset identifier MSV000100600. The mass spectrometry proteomics data have been uploaded to the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD066664. This work was supported by the National Science Centre, Poland OPUS grant UMO- 2022/45/B/NZ3/00476.嘌呤能信号通路（purinergic signaling）已成为造血过程中至关重要的调控网络，可影响造血干/祖细胞（hematopoietic stem/progenitor cells, HSPCs）的迁移与增殖。其核心嘌呤能介质为三磷酸腺苷（adenosine triphosphate, ATP）：ATP由细胞释放至造血微环境后成为细胞外ATP（extracellular ATP, eATP），作为信号配体结合P2X嘌呤能受体家族，进而促进HSPCs的迁移与增殖。随后，eATP可通过两种细胞表面表达的胞外核苷酸酶CD39与CD73在细胞外间隙降解为代谢产物细胞外腺苷（extracellular adenosine, eAdo），后者通过负反馈机制拮抗eATP的正向调控作用。 我们的研究发现，小鼠与人类HSPCs可高表达三种结合eATP的功能性P2X受体——P2X1、P2X4及P2X7，同时也高表达两种针对eAdo的P1受体——A2A与A2B。基于预实验数据，我们提出如下假说：当eATP结合P2X受体并激活胞内模式识别受体（pattern recognition receptor, PPR）Nlrp3炎症小体（Nlrp3 inflammasome）时，eAdo通过P1受体介导的信号通路则会对该模式识别受体产生相反的抑制作用。 我们的最新研究数据表明，在所谓的“毒物兴奋效应区间（hormetic zone）”即有益的生理性激活范围内，Nlrp3可促进HSPCs的迁移与增殖；该过程可被活性氧（reactive oxygen species, ROS）激活，并被血红素加氧酶-1（heme oxygenase-1, HO-1）抑制。eATP刺激细胞可使胞内ROS水平升高，而eAdo则可诱导胞质内HO-1水平上调。因此，在本研究计划中，我们拟深入探究正向（"阴"）P2受体介导与负向（"阳"）P1受体介导的信号通路对Nlrp3炎症小体调控HSPCs迁移的影响。我们将采用小鼠与人类HSPCs、嘌呤能受体缺陷型小鼠品系开展实验，并使用针对目标通路的小分子调节剂进行干预。同时，我们将借助前沿组学（OMICS）策略，深入分析mRNA与蛋白质水平的分子变化。本研究将助力优化干细胞动员与归巢方案，实现HSPCs更高效的体外扩增。 本研究涉及的数据集可于在线公共存储库中获取，存储库名称及登录号详见下文：https://www.ncbi.nlm.nih.gov/genbank/, PRJNA1128409 本研究的质谱脂质组学数据集已上传至MassIVE仓库，数据集标识符为MSV000100600。 本研究的质谱蛋白质组学数据已通过PRIDE合作仓库提交至ProteomeXchange联盟，数据集标识符为PXD066664。 本研究得到波兰国家科学中心OPUS项目（项目编号UMO-2022/45/B/NZ3/00476）的资助。