PPARα Agonists and TGFβ Inhibitors Stimulate Red Blood Cell Production by Enhancing Self-Renewal of BFU-E Erythroid Progenitors

The production of red blood cells (RBCs) in mammals is tightly regulated by erythropoietin (Epo), which stimulates erythropoiesis by promoting survival, proliferation, and terminal differentiation of the colony-forming unit erythroid progenitors (CFU-Es). Several acute and chronic anemias, including hemolysis, severe trauma-induced anemia and genetic bone marrow failure disorders such as Diamond-Blackfan anemia (DBA), are not treatable with Epo because the CFU-E erythroid progenitors that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain adequate RBC production. Treatment of Epo-resistant anemias requires a drug that acts earlier than Epo in erythropoiesis and that enhances the formation of CFU-Es. One attractive approach is to devise strategies to promote self-renewal of the upstream burst-forming unit erythroid progenitors (BFU-Es). We recently showed that, like transient amplifying cells in other stem-cell rooted developmental pathways, BFU-Es can undergo a limited number of self- renewal divisions before entering a differentiation pathway leading to formation of CFU-Es. Over time BFU-E self-renewal increases the number of CFU-Es and, after ~2-3 additional days, results in increased numbers of reticulocytes produced from each initiating BFU-E. We showed that glucocorticoids increase the probability that a BFU-E undergoes a self-renewal division and over time increase the numbers of red cells formed from a BFU-E. Recently we showed that activation of two other nuclear receptor/transcription factors - PPARα (by the lipid - lowering drugs fenofibrate and GW7647) and HIF1α (by two clinically- tested Prolyl Hydroxylase 2 (PH2) inhibitors) synergize with low corticosteroid concentrations to further enhance BFU-E self-renewal and red cell production. "Early" BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do those forming small BFU-E colonies. In order to understand the mechanism underlying this heterogeneity, we conducted single cell transcriptome analysis on BFU-E cells purified from mouse embryos. Our analyses showed that there are two principal subgroups of mouse BFU-E cells and that expression of the Type III TGFβ receptor (TGFβ RIII) is markedly elevated in "late" relative to "early" BFU-Es. Expression of TGFβ RIII is correlated with that of GATA1, a gene encoding an erythroid transcription factor induced during the BFU-E to CFU-E transition. Both mouse and human BFU-E sub populations (TGFBR3^10%lo) expressing the 10% lowest amount of surface TGFβ RIII are indeed enriched for early BFU-Es, and are significantly more responsive to glucocorticoid stimulation, which promotes BFU-E self-renewal, as compared to the total BFU-E population. The TGFBR3^10%lo BFU-E subpopulation presumably represents earlier BFU-Es with maximal capacity for self-renewal. Consistent with this notion, signaling by the TGFβ receptor kinases RI and RII increases during the transition from early (TGFBR3^10%low) to late (TGFBR3^10%hi) BFU-Es and then decreases in CFU-E cells. Blocking TGFβ signaling by receptor kinase inhibitors increases TGFBR3^10%lo BFU-E cell self-renewal and increases total erythroblast production, suggesting the use of this type of drug in treating EPO unresponsive anemias.