Foxo3a Is Essential for the Quiescence and Self-Renewal of Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state in bone marrow (BM). Quiescent stem cells show resistance to various stresses, suggesting that mechanisms for protection of HSC life from stress contribute to maintenance of self-renewal capacity through a whole life in animals. We hypothesized that a signaling pathway for regulating aging might be involved in stem cell functions.

FOXO transcription factors belong to the forkhead family of transcriptional regulators characterized by a conserved DNA-binding domain termed “forkhead box”. In C.elegans, genetic analyses have revealed the existence of a conserved insulin-like signaling involved in longevity. Conservation of this pathways lead to speculation that forkhead transcriptional factor are involved in life span in mammals. It was known that active-state Foxo3a is localized in nucleus, and we found HSC-specific nuclear localization of Foxo3a by immunocytochemistric study, therefore we generated gene-targeted Foxo3a^−/− mice to analyze roles of Foxo in HSC regulation. Peripheral blood count showed decreased number of red blood cells in Foxo3a^−/− mice, but numbers of white blood cells and platelets were normal. In colony-forming assay, we detected the numbers and sizes of myeloid, erythroid and mixed colonies derived from Foxo3a^−/− BM mononuclear cells were all normal. These results suggest that the proliferation and differentiation of Foxo3a^−/− progenitors were normal. However, the number of colony-forming cells present in long-term culture of Foxo3a^−/− c-kit⁺Sca-1⁺Lin⁻ (KSL) cells with stroma was significantly reduced. The ability of Foxo3a^−/− HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired, indicating that self-renewal capacity of HSCs was defective in Foxo3a^−/− mice. To understand the mechanisms of this phenotypes, we evaluated the cell cycle status using BrdU (5-bromodeoxyuridine) incorporation but found no difference in Foxo3a^+/+ and Foxo3a^−/− progenitor cells. To directly evaluate HSC quiescence in Foxo3a^−/− mice, we stained CD34⁻KSL cells with Pyronin Y. Although most Foxo3a^+/+ CD34⁻KSL cells stained negatively for Pyronin Y, a sizable Pyronin Y⁺ population was detected among Foxo3a^−/− CD34⁻KSL cells, demonstrating that loss of Foxo3a leads to a defect in the maintenance of HSCs quiescence. Since p38MAPK is selectively activated by environmental stress, we evaluated the activation status of p38MAPK in Foxo3a^+/+ and Foxo3a^−/− HSCs. Frequency of phosphorylated p38MAPK⁺ cells in Foxo3a^−/−CD34⁻KSL cells was significantly increased than that of Foxo3a^+/+CD34⁻KSL cells. Our results suggest that Foxo3a^−/− HSCs subjected to tangible stress in vivo. Finally, we investigated the sensitivity of Foxo3a^−/− mice to weekly 5-fluorouracil treatment in vivo. Although 60% of Foxo3a^+/+mice survived for at least 4 weeks post-injection, all Foxo3a^−/− mice were dead in 4 weeks. It suggests that Foxo3a protects hematopoietic cells from destruction by cell cycle-dependent myelotoxic agent. Taken together, our results demonstrate that Foxo3a plays a pivotal role in maintaining HSC quiescence and stress resistance.