Metabolic Pathways Control Normal and Beta-Thalassemic Erythroid Cell Maturation

Abstract 369

Erythroid cell maturation requires the integration of erythropoietin receptor (EpoR)-mediated signaling pathways and transcriptional programs of erythroid cell proliferation and differentiation largely orchestrated by GATA-1 and its transcriptional partners. Although red blood cells (RBC) purely rely on glycolysis for energy production, and metabolic processes specifically autophagy have been implicated in erythroid maturation, the potential involvement of metabolic pathways in the control of erythroid cell production is not known. Foxo3 transcription factor is a direct target of GATA-1, functionally regulated by EpoR signaling and essential for the redox regulation of erythropoiesis. Mammalian target of rapamycin (mTOR) kinase is a critical regulator of metabolic processes. We have found that generation and cycling of early erythroid precursors is controlled by a redox-mediated Foxo3-mTOR signaling. Terminal erythroid maturation is specifically compromised in Foxo3-deficient mice. Terminal maturation involves nuclear condensation, enucleation, mitochondrial clearance and complete adoption (or conversion) of glycolytic pathway by erythroid cells. We found RBC to be significantly decreased in Foxo3 mutant bone marrow and peripheral blood. This was in contrast to the increase of polychromatophilic erythroblasts associated with an increase in the total TER119⁺ population in Foxo3 mutant bone marrow likely reflecting a compensatory mechanism. Notably, using DRAQ5, a DNA-binding fluorescent dye, we found the rate of erythroblasts' enucleation in Foxo3 mutant mice to be significantly compromised. During erythroid maturation Riok3 and Mxi1 transcripts encoding for two important regulators of fetal liver erythroid enucleation are highly upregulated in the bone marrow and robustly expressed in the adult normoblasts and reticulocytes. In agreement with defective enucleation expression of both Riok3 and Mxi1 is highly reduced in Foxo3 mutant erythroblasts and reticulocytes. Interestingly, a two-week in vivo treatment of wild type (WT) and Foxo3^−/− mice with rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1) activity, increased significantly the rate of enucleation in a Foxo3-dependent manner, suggesting that mTOR requires Foxo3 activity in supporting erythroid cell maturation. Importantly, targeting mTOR ameliorates beta-thalassemia as inhibition of mTOR signaling by rapamycin treatment improved erythroid cell maturation in the bone marrow, resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase) as well as significant reduction in reticulocyte production of beta-thalassemic intermedia (th3/+) mice. Combination of thiazole orange (an RNA and DNA probe) with DRAQ5 determined that in addition to enucleation, the relative production of reticulocytes is also decreased significantly in Foxo3 mutant bone marrow. Strikingly, 6,1% of RBC (CD71 negative) in the peripheral blood of Foxo3^−/− animals contained mitochondria (CD71⁻Mito⁺) as compared to 0.7 % of wild type RBC. Autophagy is strongly implicated in late stage erythroid cell maturation and mitochondrial removal from reticulocytes. In agreement with a function for Foxo3 in control of mitochondrial removal, expression of Ulk1 (Atg1) and Nix (Bnip3l) both regulators of mitochondrial clearance via autophagy was highly downregulated in Foxo3 mutant normoblasts and reticulocytes. The expression of Nix was notable since Nix was upregulated over 40 fold in wild type but not in Foxo3 mutant reticulocytes as evaluated by the Fluidigm™ microfluidics array technology. These results are consistent with the expression pattern of Foxo3 that is highly upregulated with erythroid maturation and is the most highly expressed in normoblasts. Collectively our results indicate that Foxo3 has a key function in the regulation of terminal erythroid cell maturation. They also suggest that rapamycin may be considered for the treatment of beta-thalassemia. These results are consistent with the model of FOXO3a induction during late human erythroid cell maturation. Based on these studies we propose that Foxo3 coordinates metabolic pathways with the transcriptional program of terminal erythroid cell maturation. Understanding this metabolic program is likely to impact efficient RBC production in culture.