**6. Conclusion**

In spite of its original identification as a cellular homolog of a viral oncogene, pathophysiological roles of the Cbl family proteins remained unclear for some time. Genetic studies in model organisms as well as identification of *CBL* mutations in patient-derived specimen played crucial roles in deciphering their essential functions as regulators of HSC homeostasis. Combined with molecular/biochemical information gathered over the last two decades, we now appreciate the complexity of the regulatory pathways surrounding the Cbl family proteins. While the primary focus of studies in the last ten years has been on Cbl's E3 ubiquitin ligase functions towards phosphotyrosine motif-containing targets, observations in cells expressing mutant Cbl proteins began to challenge this relatively-simplistic viewpoint. Further studies into this multifaceted protein family may uncover opportunities for novel diagnostics and therapeutics.

#### **7. Acknowledgment**

Works in the author's laboratory is supported by grants from the US Department of Defense Breast Cancer Research Program (W81XWH-10-1-0740) and the Nebraska Department of Health and Human Services (Stem Cell 2011-06).

#### **8. References**

122 Advances in Hematopoietic Stem Cell Research

As is clear from the list of potential Cbl upstream receptors, Kit and Flt3 may not be the only targets of Cbl-dependent regulation in HSCs. Although pathways other than Kit or Flt3 have not been as carefully examined in relation to Cbl, existing evidences suggest that following

Tie2, encoded by the *TEK* gene, is an RTK expressed predominantly on endothelial cells, but they also provide crucial functions in the maintenance of quiescence and self-renewal capacity of the HSCs (Arai et al., 2004). The interaction between Tie2 and angiopoietin-1 (Ang-1), its ligand, has been shown to promote ubiquinylation of Tie2 by Cbl and receptor internalization (Wehrle et al., 2009). Structurally, the cytoplasmic domain of Tie2 does not contain any tyrosine residues that match the canonical Cbl(TKB) recognition sequence. However, activated Tie2 is known to bind Grb2 (Huang et al., 1995), thus may interact with

Cytokines such as hematopoietic growth factors and interleukins play essential roles in hematopoiesis. Cbl becomes tyrosine phosphorylated upon stimulation through various cytokine receptors (Table 2), and hematopoietic cells deficient in Cbl activity show enhanced sensitivity to cytokines (Rathinam et al., 2008; Sanada et al., 2009; Naramura et al., 2010). Receptors for these factors do not possess cytoplasmic tyrosine kinases but they activate the Janus kinase/Signal Transducers and Activators of Transcription (JAK/STAT) pathway (Yoshimura, 2009). Ligand binding induces receptor oligomerization, which activate associated JAK kinases and they, in turn, phosphorylate the receptor cytoplasmic domains

There is no solid experimental evidence supporting the direct interaction between the JAK/STAT pathway and Cbl. Activation of the JAK/STAT pathway induces the expression of Suppressor of Cytokine Signaling (SOCS) family proteins, which function as E3 ubiquitin

In addition to the JAK/STAT pathway, ligand binding to cytokine receptors activate the Ras-MAPK pathway through adaptor proteins such as APS and Grb2. Activation of this pathway is required for cell proliferation. As discussed above, these adaptor proteins are know to interact with Cbl, providing a potential link between the cytokine pathway and the

In spite of its original identification as a cellular homolog of a viral oncogene, pathophysiological roles of the Cbl family proteins remained unclear for some time. Genetic studies in model organisms as well as identification of *CBL* mutations in patient-derived specimen played crucial roles in deciphering their essential functions as regulators of HSC homeostasis. Combined with molecular/biochemical information gathered over the last two decades, we now appreciate the complexity of the regulatory pathways surrounding the Cbl

pathways may be regulated by the Cbl family proteins either directly or indirectly.

**5. Other potential targets** 

Cbl indirectly through this adaptor.

and create binding sites for SH2-containing proteins.

**5.2 Cytokine receptors** 

ligases for this pathway.

Cbl family proteins.

**6. Conclusion** 

**5.1 Tek** 


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**7** 

Keiyo Takubo

*Japan* 

**The Hypoxia Regulatory System in** 

*Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology,* 

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

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

functional effects of BM hypoxia on HSCs *in vivo* have not been fully elucidated.

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

**1. Introduction** 

**Hematopoietic Stem Cells** 

*Keio University School of Medicine, Tokyo,* 

