Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Mammalian HSCs are kept quiescent in the endosteal niche, a hypoxic zone of the bone marrow (BM). In this study, we show that normal HSCs maintain intracellular hypoxia and stabilize hypoxia-inducible factor-1alpha (HIF-1alpha) protein. In HIF-1alpha-deficient mice, the HSCs lost their cell cycle quiescence and HSC numbers decreased during various stress settings including bone marrow transplantation, myelosuppression, or aging, in a p16(Ink4a)/p19(Arf)-dependent manner. Overstabilization of HIF-1alpha by biallelic loss of an E3 ubiquitin ligase for HIF-1alpha (VHL) induced cell cycle quiescence in HSCs and their progenitors but resulted in an impairment in transplantation capacity. In contrast, monoallelic loss of VHL induced cell cycle quiescence and improved BM engraftment during bone marrow transplantation. These data indicate that HSCs maintain cell cycle quiescence through the precise regulation of HIF-1alpha levels.

Cell IntroductionQuiescence maintains the "stemness" of hematopoietic stem cells (HSCs) by protecting them from differentiation or senescence. 1 Various strategies have been used to maintain and amplify HSCs, 2-4 such as HoxB4 up-regulation, Bmi1 overexpression, Angptl addition, and Wnt3a supplementation. However, a novel strategy for the maintenance/amplification of HSCs is needed because effective expansion methodologies have not been established.F-box and WD-40 domain protein 7 (Fbxw7) is an F-box protein component of an stem cell factor-type ubiquitin ligase that contributes to ubiquitin-dependent degradation of cell-cycle activators and oncoproteins. 5 Known substrates of Fbxw7 include cyclin E, 5,6 c-Myc, 5,6 Notch1, 5,6 and mTOR, 7 which are reportedly important for G 0 state maintenance in HSCs. [8][9][10] Deletion of Fbxw7 in adult HSCs results in loss of quiescence in HSCs, defective bone marrow transplantation (BMT) capacity, and the emergence of T-cell acute lymphoblastic leukemia.In this study, Fbxw7␣ was identified as the major Fbxw7 isoform in HSCs. Fbxw7␣ overexpression in HSCs maintained cell-cycle quiescence and supported ex vivo HSC transplantation capacity. These results identify Fbxw7␣ as a key factor for HSC maintenance. Methods Retrovirus transductionFbxw7␣, ␤, and ␥ cDNAs were amplified and subcloned into the pMY-IRES-EGFP retroviral vector. 11 Production and transduction of retrovirus were performed as previously described. 12 At 48 hours after transduction, enhanced green fluorescence protein-positive (EGFP ϩ ) cells were sorted for assays. All experiments were approved by the Animal Care and Use Committee in Keio University School of Medicine. Cell-cycle analysisFor cell-cycle analysis by bromodeoxyuridine (BrdU) assay, EGFP or Fbxw7␣ virus-transduced lineage marker (Lin) Ϫ Sca-1 ϩ c-Kit ϩ (LSK) cells were cultured on fibronectin-coated plates in serum-free medium (SF-O3) containing 1.0% bovine serum albumin, 100 ng/mL murine stem cell factor, and 100 ng/mL human thrombopoietin. Cells cultured for 42 hours were labeled for 3 hours with 10M BrdU. EGFP ϩ cells were isolated and stained with anti-BrdU antibody followed by secondary antibody and TOTO-3, and the percentage of BrdU ϩ cells was calculated. Statistical analysisP values were calculated using 2-tailed unpaired Student t tests (for normal distribution), Wilcoxon tests (for non-normal distribution) for 2-group experiments, or Tukey multiple comparison tests (for multiple-group experiments).Additional procedures can be found in the supplemental data (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Results and discussionThe Fbxw7 locus encodes 3 mRNA isoforms (Fbxw7␣, ␤, and ␥), each of which has a unique 5Ј exon and 10 shared exons. Fbxw7␣

The interaction with bone marrow (BM) plays a crucial role in pathophysiological features of multiple myeloma (MM), including cell proliferation, chemoresistance, and bone lesion progression. To characterize the MM-BM interactions, we utilized an in vivo experimental model for human MM in which a GFP-expressing human MM cell line is transplanted into NOG mice (the NOG-hMM model). Transplanted MM cells preferentially engrafted at the metaphyseal region of the BM endosteum and formed a complex with osteoblasts and osteoclasts. A subpopulation of MM cells expressed VE-cadherin after transplantation and formed endothelial-like structures in the BM. CD138+ myeloma cells in the BM were reduced by p53-dependent apoptosis following administration of the nitrogen mustard derivative bendamustine to mice in the NOG-hMM model. Bendamustine maintained the osteoblast lining on the bone surface and protected extracellular matrix structures. Furthermore, bendamustine suppressed the growth of osteoclasts and mesenchymal cells in the NOG-hMM model. Since VE-cadherin+ MM cells were chemoresistant, hypoxic, and HIF-2α-positive compared to the VE-cadherin− population, VE-cadherin induction might depend on the oxygenation status. The NOG-hMM model described here is a useful system to analyze the dynamics of MM pathophysiology, interactions of MM cells with other cellular compartments, and the utility of novel anti-MM therapies.