PROJECT SUMMARY Healthy adults produce 2 million erythrocytes every second through the process of basal erythropoiesis. Insufficient erythrocyte production causes anemia, which is a significant global human health problem. During regeneration or “stress erythropoiesis” to recover from anemia, erythrocyte production further increases. Stress erythropoiesis is critical for recovery from surgery, chemotherapy, bone marrow transplantation and infection; however, the underlying mechanisms remain unclear. Both basal and stress erythropoiesis are regulated by erythropoietin (Epo) and its receptor EpoR. Epo binding to the EpoR activates the associated tyrosine kinase JAK2, which initiate downstream signaling leading to survival, proliferation and differentiation of erythroid cells. While EpoR signaling in more differentiated erythroid precursors has been extensively studied, EpoR signaling in early progenitors has not been elucidated. This proposal is based on our recent findings of a previously unrecognized population of early colony-forming erythroid progenitors (CFU-E), which we named stress CFU-E or sCFU-E. sCFU-E are targets of Epo, are only expanded in erythroid stress, and are essential for recovery of the erythron. sCFU-E are also hijacked by the activating JAK2 mutant, JAK2(V617F), to drive erythrocytosis in myeloproliferative neoplasms. In preliminary studies, we discovered that sCFU-E proliferation and differentiation involve novel Epo/EpoR signaling. We found that Epo induces expression of Nocturnin, a NADP(H) phosphatase that regulates cellular oxidative stress to promote sCFU-E proliferation. Epo also induces expression of SLC23A2, an ascorbate (Vitamin C) transporter, in sCFU-E. sCFU-E accumulates high levels of ascorbate, and ascorbate accelerates sCFU-E differentiation. The goal of this proposal is to elucidate the mechanism by which Nocturnin and ascorbate drive proliferation and differentiation of sCFU-E in the context of stress erythropoiesis and erythrocytosis. Aim 1 will determine the role of ascorbate import on sCFU-E function. Aim 2 will elucidate mechanisms underlying ascorbate-dependent sCFU-E differentiation. Aim 3 will determine the role of Nocturnin and oxidative stress signaling in sCFU-E proliferation. These results will elucidate mechanisms controlling stress erythropoiesis and erythrocytosis, and may lead to novel therapeutic interventions for anemia and those that can target erythrocytosis while preserving basal erythropoiesis.