Foxo3 Transcription Factor Regulates Oxidative Stress in In Vivo Erythropoiesis.

Reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxides are byproducts of normal oxidative metabolism that play an important role in regulating the proliferation of many cell types including hematopoietic cells. However, excess production of ROS induces damage to cellular components including DNA, maintains cellular transformation, apoptosis and senescence, and is associated with leukemias. Therefore highly regulated mechanisms are required for balancing ROS production during normal hematopoiesis. In particular, erythroid cells require an efficient scavenging mechanism for ROS because of high exposure to oxidative stress as a result of an abundance of heme iron. Specifically, deficiencies in erythroid ROS scavenging enzymes are associated with malignancies and other hematopoietic disorders such as sideroblastic anemia. We have recently shown that Erythropoietin (Epo)-induced activation of the PI3-kinase/AKT signaling pathway supports differentiation and maturation of primary erythroid progenitor cells (Zhao et al., Blood 2006; Ghaffari et al., Blood 2006). FOXO forkhead transcription factors constitute one of downstream targets of the PI3-kinase/AKT signaling pathway. In response to Epo, activation of PI3-kinase/AKT signaling pathway results in rapid phosphorylation of FOXO proteins triggering its translocation to the cytosol where, away from their transcriptional targets, FOXO are inhibited. FOXO proteins play many fundamental functions including protecting cells against oxidative insults by controlling the expression of ROS scavenging enzymes manganese superoxide dismutase and catalase. Here we show that Foxo3 is the most abundant FOXO protein in erythroid cells whose expression is upregulated several fold during maturation of mouse primary fetal liver erythroid progenitor cells. Using immunofluorescent staining and reporter gene assay, we show that Foxo3 enters the nucleus and becomes transcriptionally active as fetal liver erythroblasts mature. Notably, Foxo3 activation is concomitant with downregulation of Epo receptor (EpoR) on maturing erythroblasts. We further demonstrate that Foxo3 regulates levels of ROS in adult erythroid cells. In particular, eight to twelve week old Foxo3-deficient mice (kindly provided by Dr. Ron DePinho, Harvard Medical School) exhibit hemolysis that is responsive to in vivo therapy by N-Acetyl- l-Cysteine, a generic scavenger of ROS, suggesting that Foxo3−/ − hemolysis is induced by ROS. In addition, Foxo3−/ − hemolysis is characterized by enhanced concentration of erythroid ROS as determined by dichlorofluorescein assay and significantly decreased expression of ROS scavenging enzymes, leading to protein oxidation and shortened erythrocyte lifespan. In agreement with these results, Foxo3−/ − mice are highly sensitive to oxidative stress and, in contrast to their wild type counterparts, do not survive treatment with phenylhydrazine which reacts with hemoglobin to generate ROS. These results demonstrate that Foxo3 is required for protecting erythroid cells from oxidative stress. Finally, our data further support an important function for Foxo3 in coordinating cell cycle and differentiation of primary erythroblasts that is mediated by a ROS-regulated mechanism. Collectively, these findings establish Foxo3, a transcription factor targeted by EpoR signaling, as a regulator of oxidative stress in primary erythropoiesis.