**4. Hypoxia response system in HSCs**

Cells sense, respond, and adapt to hypoxia using hypoxia-responsive regulatory pathways. HSCs utilize the same hypoxia response pathways as a number of other cell types. A central component of these pathways is hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for cellular and systemic responses to a low oxygen microenvironment (Semenza, 2010) (Figure 3). HIF-1 is a heterodimeric transcription factor consisting of the oxygen-dependent HIF-1 subunit and an oxygen-independent HIF-1 subunit (Wang and Semenza, 1995). HIF-1 is hydroxylated at proline (Pro) 402 and/or 564 in the oxygendependent degradation (ODD) domain under normoxic conditions (Kaelin and Ratcliffe, 2008). HIF-1 is hydroxylated by three prolyl hydroxylases (PHD1–3) which require molecular oxygen, Fe2+, 2-oxoglutarate, and ascorbic acid for their full enzymatic activity (Epstein et al, 2001). Prolyl-hydroxylated HIF-1 protein is recognized by the von Hippel-Lindau (VHL) tumor suppressor protein, which recruits the Elongin C/Elongin B/Cullin2/E3 ubiquitin ligase complex. As a result, prolyl-hydroxylated HIF-1 protein is ubiquitinated and degraded by the proteasome. Under a hypoxic environment, prolyl hydroxylases lose their enzymatic activity. Thus, prolyl hydroxylation of HIF-1 is suppressed, and HIF-1 protein is stabilized without degradation (Kaelin and Ratcliffe, 2008). HIF-1 heterodimers (HIF-1:HIF-1) are recruited and bind to hypoxia response elements (HREs) in various target genes and activate transcription programs (Semenza, 2010).

lapse and three-dimensional observation with a multi-photon microscope) of single LT-HSCs in the BM has shed light on this paradox. Real-time tracking of murine BM revealed that HSCs gradually move away from bone marrow blood vessels and then detach from them and translocate to the osteoblastic zone of the BM after transplantation (Lo Celso et al, 2009; Xie et al, 2009). Based on these observations, it is possible that subpopulations of HSCs residing in different specific locations have different

In parallel with the hypoxic microenvironment for HSCs *in vivo*, hypoxic culture phenotypically and functionally sustained HSCs more effectively than normoxic culture (20% oxygen). Also, hypoxic culture enhances the colony-forming ability (progenitor ability) and transplantation capacity (HSC capacity) of cultured BM cells or isolated HSCs (Cipolleschi et al, 1993; Danet et al, 2003; Ivanovic et al, 2004). Hypoxic treatment also induces cell cycle quiescence in cultured HSC (Hermitte et al, 2006; Shima et al, 2010). Quiescent HSCs are defined by a high amount of efflux of the DNA-binding dye Hoechst 33342 from the cytosol (Goodell et al, 1996). These cells are called "side population (SP)" cells due to their specific staining pattern by flow cytometric analysis. Hypoxic treatment also sustains the SP phenotype in HSC *in vitro* (Krishnamurthy et al, 2004). Exclusion of Hoechst dye from the HSC cytosol is supported by Bcrp1/ABCG2, an ATP-dependent transporter, at the plasma membrane. When HSCs were cultured under hypoxic conditions, mRNA expression of Bcrp1/ABCG2 was significantly increased and the number of SP cells was also increased as compared to HSCs cultured at normoxia. Interestingly, because Bcrp1- /- mice show no significant defect in hematopoiesis (Zhou et al, 2001), the functional role of

Cells sense, respond, and adapt to hypoxia using hypoxia-responsive regulatory pathways. HSCs utilize the same hypoxia response pathways as a number of other cell types. A central component of these pathways is hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for cellular and systemic responses to a low oxygen microenvironment (Semenza, 2010) (Figure 3). HIF-1 is a heterodimeric transcription factor consisting of the oxygen-dependent HIF-1 subunit and an oxygen-independent HIF-1 subunit (Wang and Semenza, 1995). HIF-1 is hydroxylated at proline (Pro) 402 and/or 564 in the oxygendependent degradation (ODD) domain under normoxic conditions (Kaelin and Ratcliffe, 2008). HIF-1 is hydroxylated by three prolyl hydroxylases (PHD1–3) which require molecular oxygen, Fe2+, 2-oxoglutarate, and ascorbic acid for their full enzymatic activity (Epstein et al, 2001). Prolyl-hydroxylated HIF-1 protein is recognized by the von Hippel-Lindau (VHL) tumor suppressor protein, which recruits the Elongin C/Elongin B/Cullin2/E3 ubiquitin ligase complex. As a result, prolyl-hydroxylated HIF-1 protein is ubiquitinated and degraded by the proteasome. Under a hypoxic environment, prolyl hydroxylases lose their enzymatic activity. Thus, prolyl hydroxylation of HIF-1 is suppressed, and HIF-1 protein is stabilized without degradation (Kaelin and Ratcliffe, 2008). HIF-1 heterodimers (HIF-1:HIF-1) are recruited and bind to hypoxia response elements (HREs) in various target genes and activate transcription programs (Semenza,

oxygenation statuses.

Bcrp1 in HSCs is still uncharacterized.

2010).

**4. Hypoxia response system in HSCs** 

The diagrams represent the regulation of HIF-1 protein and interacting factors under different oxygen conditions. HIF-1 is a substrate for both prolyl and asparaginyl hydroxylases. Under normoxia, proline and asparagine residues are hydroxylated. These modifications regulate the stability and transcriptional activity of HIF-1. bHLH, basic-helix-loop-helix domain; PAS, Per-ARNT-Sim domain; TAD-N, transactivation domain N-terminal; ID, inhibitory domain; TAD-C, C-terminal transactivation domain; PHD, prolyl hydroxylase domain-containing protein; and FIH-1, factor-inhibiting HIF-1.

#### Fig. 3. Regulation of hypoxia-inducible factor-1 (HIF-1)

The Hypoxia Regulatory System in Hematopoietic Stem Cells 141

HIF-1 mRNA and protein are highly expressed in LT-HSCs (Takubo et al, 2010; Simsek et al, 2010) (Figure 4). HSCs derived from conditional HIF-1 knockout (HIF-1) mice have a defective capacity for marrow reconstitution during serial BM transplantation (Takubo et al., 2010). HIF-1 LT-HSCs lost cell cycle quiescence, entered the cell cycle from G0 phase, proliferated, and showed reduced tolerance to stresses such as 5-fluorouracil administration or aging. These studies suggest that HIF-1 plays an essential role in the regulation of HSC quiescence and stress resistance *in vivo*. In addition to these HIF-1 loss-of-function studies, conditional deletion of the VHL gene in hematopoietic cells was performed as a HIF-1 gain-of-function experiment. Analysis of VHL mutant hematopoietic cells revealed that the functional properties of LT-HSCs and progenitors are differentially influenced by HIF-1. HIF-1 protein levels are elevated in either biallelic (VHL) or monoallelic (VHL+) conditional knockout hematopoietic cells. For example, only a minor population of normal hematopoietic progenitors (CD34+ LSK cells) are in a quiescent state. In clear contrast, the

higher. At steady state, HIF-1 protein levels are not high in hematopoietic progenitors, and forced stabilization of HIF-1 protein through monoallelic VHL deletion induces VHL+/ CD34+ LSK progenitors to exit the cell cycle and maintains them in the G0 phase. Severe suppression of cell cycling and transplantation capacity is restored in HIF-1:VHL doubly mutated HSCs. The decreased frequency of LT-HSCs seen in VHL mice is rescued by the co-deletion of the HIF-1 gene *in vivo*. Also, long-term *in vitro* exposure of LT-HSCs to a PHD inhibitor (dimethyloxalylglycine; DMOG), which stabilizes HIF-1α even under normoxic conditions, attenuates stem cell ability especially during BM transplantation

Collectively, these results provide evidence that there is an optimal HIF-1 protein level for HSC maintenance. HIF-1 is required for stress resistance and long-term maintenance of HSCs, and within an appropriate range, moderate increases of HIF-1 (to the level caused by VHL heterozygous deletion) are trophic for HSCs through the induction of quiescence. However, aberrantly high HIF-1 levels are also harmful to HSCs and lead to a loss of stem cell capacity and the exhaustion of the HSC pool. Homozygous deletion of VHL results in a severe suppression of the cell cycle and a homing defect during

HIF-1 not only acts in the HSC system but also plays an important role in neural stem cells (NSCs) under hypoxic conditions. In this type of cell, HIF-1 induces the activation of the Wnt/-catenin signalling pathway through the upregulation of -catenin and the expression of the downstream transcription factors lymphoid enhancer-binding factor 1 and T-cell factor 1 (Mazumdar J et al, 2010). Wnt/-catenin activity was closely correlated with hypoxic status in the subgranular zone of the hippocampus, which is one of the niches for NSCs. Loss of HIF-1 in NSCs resulted in a defective Wnt-dependent hippocampal neurogenic niche capacity. As a result, NSC proliferation and differentiation, and the production of new neurons, were attenuated. Interestingly, the biological effects of HIF-1 on NSCs (cell cycle promotion) are clearly different from those seen in HSCs (cell cycle quiescence). It will be important to dissect how these different lineage stem cell systems utilize the same protein (HIF-1) to sustain themselves using different downstream molecular machinery and biological events. It is also of interest to investigate embryonic

hematopoietic progenitors in the quiescent phase is significantly

proportion of VHL+/

(Eliasson P et al, 2010).

transplantation.

Scheme of biological outcomes of different HIF-1 protein levels in HSCs. This is achieved by HIF-1 or VHL deletion in HSCs using knockout mouse models. Normal HSCs (the second from the top) stabilize HIF-1, which maintains cell cycle quiescence at the hypoxic bone marrow niche in the endosteum. Preferential stabilization of HIF-1 was observed in HSCs under hypoxia. HIF-1 HSCs (top) lose cell cycle quiescence and stress resistance against transplantation, chemotherapeutic agents, and aging. In addition, HIF-1 HSCs leave the bone marrow niche and drive extramedullary hematopoiesis in the spleen. Production of ROS is accelerated in HIF-1 HSCs. Heterozygous deletion of VHL results in a slight increase in HIF-1 protein. Under these conditions, cell cycle quiescence in HSCs is enhanced. The VHL+/ HSC (the second from the bottom) is resistant to transplantation and aging. ROS production is also suppressed in VHL+/ HSCs. The homozygous VHL mutant (VHL) HSC has a maximal dose of HIF-1 protein. In contrast to heterozygous VHL mutant HSCs, VHL HSCs completely lost stem cell capacity potentially due to aberrant suppression of the cell cycle and/or homing capacity to the niche. This defect is HIF-1–dependent because the co-deletion of HIF-1 in homozygous VHL-deficient hematopoietic cell rescued the defect. Thus, the precise regulation of HIF-1 levels coordinates stem cell proliferation and differentiation. Recently, it has been reported that vascular endothelial growth factor, heat shock proteins, and GRP78 and its ligand Cripto regulate HSC quiescence and maintain HSCs in hypoxia as downstream factors of HIF-1 (Rehn et al, 2011; Miharada et al, 2011).

Fig. 4. Features of HIF-1 or VHL knockout HSCs

Scheme of biological outcomes of different HIF-1 protein levels in HSCs. This is achieved by HIF-1 or VHL deletion in HSCs using knockout mouse models. Normal HSCs (the second from the top) stabilize HIF-1, which maintains cell cycle quiescence at the hypoxic bone marrow niche in the endosteum. Preferential stabilization of HIF-1 was observed in HSCs under hypoxia. HIF-1 HSCs (top) lose cell cycle quiescence and stress resistance against transplantation, chemotherapeutic agents, and aging. In addition, HIF-1 HSCs leave the bone marrow niche and drive extramedullary hematopoiesis in the spleen. Production of ROS is accelerated in HIF-1 HSCs. Heterozygous deletion of VHL results in a slight increase in HIF-1 protein. Under these conditions, cell cycle quiescence in HSCs is enhanced. The

protein. In contrast to heterozygous VHL mutant HSCs, VHL HSCs completely lost stem cell capacity potentially due to aberrant suppression of the cell cycle and/or homing capacity to the niche. This defect is HIF-1–dependent because the co-deletion of HIF-1 in homozygous VHL-deficient hematopoietic cell rescued the defect. Thus, the precise regulation of HIF-1 levels coordinates stem cell proliferation and differentiation. Recently, it has been reported that vascular endothelial growth factor, heat shock proteins, and GRP78 and its ligand Cripto regulate HSC quiescence and maintain HSCs in hypoxia as downstream

HSC (the second from the bottom) is resistant to transplantation and aging. ROS production is also

HSCs. The homozygous VHL mutant (VHL) HSC has a maximal dose of HIF-1

VHL+/

suppressed in VHL+/

factors of HIF-1 (Rehn et al, 2011; Miharada et al, 2011). Fig. 4. Features of HIF-1 or VHL knockout HSCs

HIF-1 mRNA and protein are highly expressed in LT-HSCs (Takubo et al, 2010; Simsek et al, 2010) (Figure 4). HSCs derived from conditional HIF-1 knockout (HIF-1) mice have a defective capacity for marrow reconstitution during serial BM transplantation (Takubo et al., 2010). HIF-1 LT-HSCs lost cell cycle quiescence, entered the cell cycle from G0 phase, proliferated, and showed reduced tolerance to stresses such as 5-fluorouracil administration or aging. These studies suggest that HIF-1 plays an essential role in the regulation of HSC quiescence and stress resistance *in vivo*. In addition to these HIF-1 loss-of-function studies, conditional deletion of the VHL gene in hematopoietic cells was performed as a HIF-1 gain-of-function experiment. Analysis of VHL mutant hematopoietic cells revealed that the functional properties of LT-HSCs and progenitors are differentially influenced by HIF-1. HIF-1 protein levels are elevated in either biallelic (VHL) or monoallelic (VHL+) conditional knockout hematopoietic cells. For example, only a minor population of normal hematopoietic progenitors (CD34+ LSK cells) are in a quiescent state. In clear contrast, the proportion of VHL+/ hematopoietic progenitors in the quiescent phase is significantly higher. At steady state, HIF-1 protein levels are not high in hematopoietic progenitors, and forced stabilization of HIF-1 protein through monoallelic VHL deletion induces VHL+/ CD34+ LSK progenitors to exit the cell cycle and maintains them in the G0 phase. Severe suppression of cell cycling and transplantation capacity is restored in HIF-1:VHL doubly mutated HSCs. The decreased frequency of LT-HSCs seen in VHL mice is rescued by the co-deletion of the HIF-1 gene *in vivo*. Also, long-term *in vitro* exposure of LT-HSCs to a PHD inhibitor (dimethyloxalylglycine; DMOG), which stabilizes HIF-1α even under normoxic conditions, attenuates stem cell ability especially during BM transplantation (Eliasson P et al, 2010).

Collectively, these results provide evidence that there is an optimal HIF-1 protein level for HSC maintenance. HIF-1 is required for stress resistance and long-term maintenance of HSCs, and within an appropriate range, moderate increases of HIF-1 (to the level caused by VHL heterozygous deletion) are trophic for HSCs through the induction of quiescence. However, aberrantly high HIF-1 levels are also harmful to HSCs and lead to a loss of stem cell capacity and the exhaustion of the HSC pool. Homozygous deletion of VHL results in a severe suppression of the cell cycle and a homing defect during transplantation.

HIF-1 not only acts in the HSC system but also plays an important role in neural stem cells (NSCs) under hypoxic conditions. In this type of cell, HIF-1 induces the activation of the Wnt/-catenin signalling pathway through the upregulation of -catenin and the expression of the downstream transcription factors lymphoid enhancer-binding factor 1 and T-cell factor 1 (Mazumdar J et al, 2010). Wnt/-catenin activity was closely correlated with hypoxic status in the subgranular zone of the hippocampus, which is one of the niches for NSCs. Loss of HIF-1 in NSCs resulted in a defective Wnt-dependent hippocampal neurogenic niche capacity. As a result, NSC proliferation and differentiation, and the production of new neurons, were attenuated. Interestingly, the biological effects of HIF-1 on NSCs (cell cycle promotion) are clearly different from those seen in HSCs (cell cycle quiescence). It will be important to dissect how these different lineage stem cell systems utilize the same protein (HIF-1) to sustain themselves using different downstream molecular machinery and biological events. It is also of interest to investigate embryonic

The Hypoxia Regulatory System in Hematopoietic Stem Cells 143

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