Erythropoietin (Epo) signaling is required for differentiation of erythroid progenitors to mature red blood cells. Binding of Epo to its receptor activates Jak2, which in turn activates many signaling proteins including AKT, MAPK proteins and STATs. We have shown previously that AKT is required for Epo regulation of erythroid cell maturation; activated AKT complements Epo receptor signaling in JAK2-deficient fetal liver cells and supports erythroid cell differentiation. AKT functions by phosphorylating several proteins including FoxO3 and mTOR. AKT phosphorylation of FoxO3 represses FoxO3’s activity, whereas AKT-dependant phosphorylation activates mTOR and its downstream target p70 S6 kinase (S6K). We have shown recently that FoxO3 is essential for the regulation of erythroid cell cycling, maturation, lifespan and anti-oxidant response (Marinkovic et al., JCI, 2007). Here we aimed at identifying other proteins in AKT signaling network that may regulate the maturation of erythroid progenitors. To address this, we inhibited several signaling pathways and analyzed their role in Epo-dependant maturation of freshly-isolated E14 fetal liver progenitors. As anticipated, blocking PI3-Kinase resulted in 60 % reduction of BFU-E- and CFU-E-derived colony formation and blocked the maturation of erythroid progenitors. Interestingly, blocking either p38 or ERK MAPK signaling showed 40% reduction in erythroid BFU-E- and CFU-E-derived colony formation. Surprisingly, blocking of mTOR signaling inhibited the formation of BFU-E- and CFU-E-derived colonies by 75 %. Further analysis by flow cytometry monitoring of cell surface markers CD71 and TER 119 showed that erythroid progenitor cell maturation could not proceed past early erythroblast stage when cells were cultured in the presence of rapamycin overnight. We confirmed that this block in differentiation was not due to apoptosis of erythroid cells. Since both FoxO3 and mTOR work downstream of AKT, we asked whether inhibition of mTOR has any impact on FoxO3 activity. Epo stimulation of freshly isolated bone marrow lineage-negative cells previously starved from cytokines showed a 2.3 fold increase in FoxO3 phosphorylation in the presence of rapamycin, suggesting cross talk between mTOR and FoxO3. Next, we investigated the effect of loss of FoxO3 on AKT/mTOR signaling in erythroid precursors. To address this, we prepared a population of bone marrow depleted from lineage-restricted cells and cultured under optimum erythroid conditions that generated 60% erythroblasts after 18 hours. Epo stimulation of FoxO3 null erythroid precursors led to hyperphosphorylation of Jak2, AKT, mTOR and S6K as compared to control cells. Since FoxO3 is critical for repression of reactive oxygen species (ROS), we evaluated the potential role of ROS in activating these proteins in FoxO3 mutant erythroid cells. In vitro treatment with ROS scavenger N-Acetyl-Cysteine (NAC) reduced significantly the hyper-phosphorylation of AKT, mTOR and S6K in FoxO3 null erythroid precursors in response to Epo. In addition, our results suggest that phosphorylation of JAK2 and its downstream signaling proteins AKT/mTOR/S6K in primary wild type erythroid precursor cells in response to Epo is mediated by ROS. Interestingly, ROS modulation of phosphorylation of mTOR/S6K was significantly stronger than that of AKT in response to Epo-stimulation of primary erythroid cells. Activation of AKT/mTOR/S6K is likely to mediate increased production of erythroid precursors observed in FoxO3 mutant mice (Marinkovic et al., JCI, 2007). Collectively these results indicate an important function for the AKT/mTOR/S6K signaling pathway in Epo-dependant erythropoiesis and suggest that cytokine-mediated production of ROS plays a critical role in the regulation of primary erythroid cell formation.

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