Regulation of Erythroid Cell Maturation Is Mediated by a Foxo3-mTOR Cross Talk: Outcome for Beta-Thalassemic Erythropoiesis

Abstract 176

Differentiation of erythroid progenitors to mature red blood cells requires erythropoietin receptor (EpoR) signaling. Stimulation of EpoR results in Jak2-mediated activation of mainly AKT, ERK/MAPK and STAT5 signaling pathways. Although alteration of these pathways is involved with the pathophysiology of major erythroid disorders such as beta-thalassemia mechanisms by which these signals impact transcriptional programs of erythroid cell maturation are largely unknown. We have shown previously that AKT signaling is required for Epo-mediated erythroid cell maturation and identified Foxo3 transcription factor, that is negatively regulated by AKT, as a critical regulator of erythroid cell cycle, maturation and lifespan mostly through the control of oxidative stress (Marinkovic et al., JCI, 2007). In addition to Foxo3, AKT regulates several proteins including the mammalian target of rapamycin (mTOR). Here we asked how Foxo3 regulation of oxidative stress impacts erythroid cell maturation. We found that AKT/mTOR signaling pathway is constitutively activated, possibly as part of a feedback loop, in primary Foxo3^−/− erythroid precursors. In addition, Epo stimulation of primary Foxo3^−/− erythroid precursors led to hyperphosphorylation of Jak2, AKT, mTOR and its target p70S6 Kinase (S6K) as compared to control cells. Since Foxo3 controls levels of reactive oxygen species (ROS) in erythroid cells, and ROS are known to modify signaling proteins, we asked whether ROS are involved in the hyperactivation of AKT/mTOR signaling pathway in Foxo3^−/− erythroid precursors. Combined in vivo and in vitro treatment of Foxo3^−/− erythroid precursors with ROS scavenger N-Acetyl-Cysteine (NAC) reduced significantly the hyper-phosphorylation of AKT, mTOR and S6K in response to Epo. These results strongly suggest that ROS mediate the hyperactivation of AKT/mTOR signaling pathway in Foxo3^−/− erythroid precursors. Next we addressed whether the imbalanced production versus maturation of Foxo3^−/− erythroid precursors (Marinkovic et al., JCI, 2007) is due to the constitutive activation of AKT/mTOR signaling. This was indeed the case since in vivo treatment of Foxo3^−/− mice for three weeks with the mTOR inhibitor rapamycin shifted the balance from immature towards mature erythroid cells. Interestingly while rapamycin treatment decreased cycling of Foxo3^−/− erythroid progenitors as anticipated, it resulted in highly increased proliferation of Foxo3^−/− mature erythroblasts as analyzed by in vivo BrdU assay. Importantly, the described Foxo3^−/− erythroid phenotype was maintained on two distinct genetic backgrounds (C57BL/6 and BALB/c) in mice. These results strongly suggest that the oxidative stress-induced activation of mTOR signaling pathway mediates the imbalanced production of mature erythroid cells in Foxo3^−/− mice. Given that both oxidative stress and delayed erythroid cell differentiation as seen in Foxo3^−/−erythroid precursors, contribute significantly to beta-thalassemia, we asked whether the mTOR signaling is involved in the pathogenesis of this disease. Rapamycin treatment improved erythroid cell maturation in the bone marrow as analyzed by cell size, CD44, TER 119 and CD71 surface markers, and resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase), significant reduction in reticulocyte production as well as decrease in the spleen size of beta-thalassemic intermedia (th3/+) mice similar to what was seen in Foxo3^−/− mice. Collectively these results indicate an important function for the Foxo3-mTOR cross talk in the regulation of erythroid cell maturation and suggest that rapamycin may be considered for treatment of beta-thalassemia.