**1. Introduction**

132 Advances in Hematopoietic Stem Cell Research

Wisniewski, D., Strife, A., & Clarkson, B. (1996). c-kit ligand stimulates tyrosine

Wollberg, P., Lennartsson, J., Gottfridsson, E., Yoshimura, A., & Rönnstrand, L. (2003). The

Wong, A., Lamothe, B., Lee, A., Schlessinger, J., Lax, I., & Li, A. (2002). FRS2 attenuates

Yokouchi, M., Kondo, T., Houghton, A., Bartkiewicz, M., Horne, W. C., Zhang, H.,

Yoon, C. H., Lee, J., Jongeward, G. D., & Sternberg, P. W. (1995). Similarity of sli-1, a

Yoshihara, H., Arai, F., Hosokawa, K., Hagiwara, T., Takubo, K., Nakamura, Y., Gomei, Y.,

Yoshimura, A. (2009). Regulation of cytokine signaling by the SOCS and Spred family

Zeng, S., Xu, Z., Lipkowitz, S., & Longley, J. B. (2005). Regulation of stem cell factor receptor signaling by Cbl family proteins (Cbl-b/c-Cbl). *Blood*, Vol. 105, pp. 226–232. Zhang, S., Mantel, C., & Broxmeyer, H. E. (1999). Flt3 signaling involves tyrosyl-

Zheng, N., Wang, P., Jeffrey, P. D., & Pavletich, N. P. (2000). Structure of a c-Cbl-UbcH7

Tyr-936 in c-Kit. *Biochem. J*, Vol. 370, pp. 1033–1038.

*Proc. Natl. Acad. Sci. U.S.A.*, Vol. 99, pp. 6684–6689.

UbcH7. *J. Biol. Chem*, Vol. 274, pp. 31707–31712.

osteoblastic niche. *Cell Stem Cell*, Vol. 1, pp. 685–697.

Baf3/Flt3 cells. *J. Leukoc. Biol.*, Vol. 65, pp. 372–380.

c-cbl. *Science*, Vol. 269, pp. 1102–1105.

proteins. *Keio J Med*, Vol. 58, pp. 73–83.

533–539.

10, pp. 1436–1442.

phosphorylation of the c-Cbl protein in human hematopoietic cells. *Leukemia*, Vol.

adapter protein APS associates with the multifunctional docking sites Tyr-568 and

FGF receptor signaling by Grb2-mediated recruitment of the ubiquitin ligase Cbl.

Yoshimura, A., & Baron, R. (1999). Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and

regulator of vulval development in C. elegans, to the mammalian proto-oncogene

Iwasaki, H., Matsuoka, S., Miyamoto, K., et al. (2007). Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the

phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in

complex: RING domain function in ubiquitin-protein ligases. *Cell*, Vol. 102, pp.

Stem cells localize to specific sites called 'niches' in various tissues, where they are preferentially maintained by growth factors from the environment. Mammalian bone marrow (BM) has been shown to be relatively hypoxic compared to other tissues, and primitive hematopoietic cells, including hematopoietic stem cells (HSCs), are thought to localize to the most hypoxic microenvironments in the BM. The hypoxic *ex vivo* culture of BM cells or primitive hematopoietic progenitors results in the maintenance of the primitive phenotype and cell cycle quiescence (Mohyeldin et al., 2010; Suda et al., 2011). *Ex vivo* culture of human HSCs under hypoxia also stabilizes hypoxia-inducible factor-1 (HIF-1), a master transcriptional regulator of the cellular and systemic hypoxic response, and induces various downstream effectors of HIF-1 (Danet et al., 2003). However, the regulatory mechanisms and functional effects of BM hypoxia on HSCs *in vivo* have not been fully elucidated.

In the stem cell niche, HSCs are quiescent and show slow cell cycling. Various extracellular ligands, including CXCL12 (Sugiyama et al., 2006), angiopoietin-1 (Arai et al., 2004), and/or thrombopoietin (TPO) (Qian et al., 2007; Yoshihara et al., 2007), contribute to the quiescence of HSCs. Quiescent HSCs are maintained at a lower oxidative stress state to avoid their differentiation and exhaustion (Jang & Sharkis 2007). HIF-1 is a bHLH-PAS–type transcription factor (Semenza, 2007, 2009, 2010). Under normoxic conditions, prolyl residues in the HIF-1 oxygen-dependent degradation domain (ODD) are hydroxylated by HIF prolyl hydroxylases (PHDs). The hydroxylated ODD domain of HIF-1 protein is recognized by an E3 ubiquitin ligase, the von Hippel-Lindau protein (VHL). In the autosomal dominant hereditary disorder von Hippel Lindau disease, VHL is mutated, resulting in overstabilized HIF-1 protein by the impaired ubiquitin-proteasome pathway. Under hypoxic conditions, PHDs are inactivated and HIF-1 protein escapes degradation. Several niche factors, such as thrombopoietin (TPO) (Kirito et al., 2005) and stem cell factor (SCF) (Pedersen et al., 2008), also stabilize HIF-1 protein in hematopoietic cells even under normoxic conditions.

Stabilized HIF-1 protein forms a heterodimeric transcriptional complex with the oxygenindependent subunit HIF-1, translocates to the nucleus, and directly binds hypoxiaresponsive elements found in the promoter regions of numerous downstream regulators, thereby activating their transcription. HIF-1 is reportedly required for hematopoietic cell

The Hypoxia Regulatory System in Hematopoietic Stem Cells 135

(Snippert and Clevers, 2011). Therefore, quiescence itself is not the only strategy for the

Life-long hematopoiesis is maintained by long-term (LT)-HSCs and their progeny. LT-HSCs have a two cell cycle states: a quiescent state (G0 phase) and a cycling state (non-G0; i.e., G1/S/G2/M phase). LT-HSCs in the former state are resistant to various cytotoxic stresses. Reactive oxygen species (ROS) change the cell cycle state of LT-HSCs from quiescent to cycling. Cycling LT-HSCs are also promoted to differentiate into short-term (ST)-HSCs and multipotent progenitors (MPPs). These differentiated

progenitors actively produce various terminally differentiated hematopoietic cells.

Fig. 1. Quiescent and cycling hematopoietic stem cells (HSCs)

long-term maintenance of stem cells.

generation during ontogeny. However, a detailed analysis of the contribution of HIF-1to the maintenance of adult HSCs has not yet been reported.

We analyzed HSCs in HIF-1– and VHL-deficient mice and found that the cellular pool and cell cycle status of HSCs were regulated by the HIF-1 level (Takubo et al., 2010). Our analysis revealed that the regulation of the HIF-1 dose is critical for HSC maintenance in the hypoxic niche microenvironment of the BM. The critical role for HIF-1 in HSC cell cycle regulation broadens the involvement of oxygen status in the stem cell niche. It also implies a novel strategy for maintaining and expanding HSC resources based on cellular oxygen metabolism reprogramming, including the modulation of HSC quiescence through the oxygenation status of HIF-1.
