Adenosine A2B Receptor Controls Erythroid Lineage Commitment in Stress Erythropoiesis By Promoting Metabolic Reprogramming

Abstract Insufficient oxygen availability under stress conditions including hypoxia and anemia is a major stimulus for stress erythropoiesis. Adenosine is known to be induced under hypoxia and energy depletion. Increased adenosine signaling via its specific receptors regulates multiple cellular functions including anti-inflamation, anti-vascular leakage and vasodilation. However, its function in stress erythropoiesis and underlying mechanisms are enigmatic. Among four adenosine receptors, we report that adenosine A2B receptor (ADORA2B) is expressed at a significant higher level in megakaryocyte-erythroid progenitor (MEP) compared to common pluoripotent progenitors (CMP) or granulocyte-erythroid progenitor (GMP) in undifferentiated human CD34+. To determine the function role of ADORA2B in stress erythropoiesis, we generated erythroid Adora2b specific knockouts by crossing Adora2bf/fmice with EpoR-Cre+mice. First, we demonstrated that EpoR specifically ablated ADORA2B gene only in MEP but not in CMP or GMP lineages. Next, we challenged EpoR-Cre+mice (control) and Adora2bf/fEpoR-Cre+ mice (erythroid specific ablation of Adora2b genes) with hypoxia. We discovered that genetic deletion of ADORA2B at MEP stage blocked erythroid vs myeloid commitment under hypoxia-induced stress erythropoiesis. Further metabolic profiling revealed that ADORA2B activation regulated erythroid lineage commitment by promoting glucose uptake and erythroid metabolic reprogramming channelling glucose metabolism toward the pentose phosphate pathway (PPP) rather than glycolysis to generate more ribose phosphate as well as facilitate glutamine uptake to serve as a nitrogen donor for de novo nucleotide biosynthesis. Meanwhile, ADORA2B-stimulated glutaminolysis increased TCA cycle intermediates and thus increased energy production under stress erythropoiesis. We further demonstrated that ADORA2B on MEP is also important for erythroid commitment in response to PHZ-induced mouse model. Followup studies revealed that HIF-1a is induced in erythroid progenitors in a ADORA2B-dependent manner and ablation of HIF-1a only in MEP but not in CMP or GMP attenuated erythroid lineage commitment in both hypoxia-induced and anemia-induced stress erythropoiesis mouse models. Moreover, we showed that ADORA2B-triggered metabolic reprogramming depended on HIF-1a-preferentially upregulated gene expression of transporters for glucose and glutamine and key enyzmes of PPP and glutaminolysis over glycolytic enzymes. Similar to mouse studies, in cultured Epo-stimulated human CD34+ hematopoietic stem progenitor cells, enhancing ADORA2B signaling induced gene expression of the transporters for glucose and glutamine, key enzymes of PPP and glutaminolysis over glycolysis and thus controlled the commitment to erythrioid versus myeloid lineage and in turn promoted erythroid colony formation including BFU-E, CFU-E versus CFU-GM. Further studies showed that inhibition of HIF-1a by Chrysin significantly attenuated ADORA2B activation-induced upregulation of gene expression of the transporters of glucose and glutamine, metabolic enzymes and thus reduces erythroic commitment and BFU-E and CFU-E in Epo-stimualted CD34+ HPSCs. Overall, using multidisciplinary approaches including mouse genetics, metabolomics, isotopically labelled glucose and glutamine flux analysis, human CD34+ HPSCs and pharmacological studies, we provide both mouse and human evidence that ADORA2B is a missing cofactor controlling erythroid lineage commitment in stress erythropoiesis via HIF-1a-dependent upregulation of key genes to promote metabolic reprogramming. These findings add significant new insights to erythroid commitment and immediately provide new strategies for regulating stress erythropoiesis. Disclosures Nemkov: Omix Technologies inc: Equity Ownership.