Foxo3 is required for the regulation of oxidative stress in erythropoiesis
Dragan Marinkovic, … , Tara Huber, Saghi Ghaffari
Dragan Marinkovic, … , Tara Huber, Saghi Ghaffari
Published August 1, 2007
Citation Information: J Clin Invest. 2007;117(8):2133-2144. https://doi.org/10.1172/JCI31807.
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Research Article

Foxo3 is required for the regulation of oxidative stress in erythropoiesis

Abstract

Erythroid cells accumulate hemoglobin as they mature and as a result are highly prone to oxidative damage. However, mechanisms of transcriptional control of antioxidant defense in erythroid cells have thus far been poorly characterized. We observed that animals deficient in the forkhead box O3 (Foxo3) transcription factor died rapidly when exposed to erythroid oxidative stress–induced conditions, while wild-type mice showed no decreased viability. In view of this striking finding, we investigated the potential role of Foxo3 in the regulation of ROS in erythropoiesis. Foxo3 expression, nuclear localization, and transcriptional activity were all enhanced during normal erythroid cell maturation. Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of p21CIP1/WAF1/Sdi1 (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role.

Authors

Dragan Marinkovic, Xin Zhang, Safak Yalcin, Julia P. Luciano, Carlo Brugnara, Tara Huber, Saghi Ghaffari

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Figure 4

Foxo3 expression is upregulated during erythroblast maturation.

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Foxo3 expression is upregulated during erythroblast maturation. (A) QRT-...
(A) QRT-PCR analysis of Foxo1, Foxo3, and Foxo4 in embryonic and adult hematopoietic organs. Shown are representative results from 3 independent experiments performed in duplicate. (B) Western blot analysis of endogenous FoxO protein expression in the bone marrow. Protein lysates of HEK293T cells overexpressing FOXO1, FOXO3a, and FOXO4 cDNAs were used as positive controls. (C) QRT-PCR analysis of indicated transcripts from subpopulations of fetal liver isolated by flow cytometry according to their CD71 and TER119 expression. Note that expression of Foxo3 is the highest in the most mature erythroid cell subpopulation (CD71–TER119+). Representative results from 3 independent experiments performed in triplicate are shown as mean ± SEM. Cells differentiate from a double CD71–TER119– cell subpopulation enriched in hematopoietic progenitors, including erythroid progenitors, to CD71+TER119– cells containing mostly proerythroblasts and basophilic erythroblasts, then to CD71+TER119+ cells enriched for basophilic/polychromatophilic erythroblasts, and finally to CD71–TER119+ cells consisting mostly of normoblasts. (D) Western blot analysis of Foxo3 in subpopulations of fetal liver enriched for progenitors (TER119–) and for erythroid precursors (TER119+, erythroblasts) using anti-FOXO3a antibody. Anti–GATA-1 (N6) and anti–β-actin antibodies were used as controls.