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.

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1␣ gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1␣ influenced p53, we also created double-knockout (HIF-1␣ null, p53 null) strains and cells. In both cell types, loss of HIF-1␣ abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1␣ genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1␣ causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1␣-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1␣ dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.Mammalian cells have evolved to utilize molecular oxygen for energy production. Cells can respond differently to wide ranges of oxygen through alterations in both their metabolic states and growth rates. In recent years, several lines of evidence have indicated that hypoxia can alter cell proliferation in two distinct ways: via programmed cell death and through growth arrest. In transformed cells, hypoxia can provoke apoptosis via the p53 pathway; ultimately, this can represent a potent mechanism for the selection of p53 mutants in tumor cell populations (30,34). Nontransformed hypoxic cells, on the other hand, can undergo cell cycle arrest at the G 1 /S interface without any alteration in their long-term viability (10).It has been proposed that hypoxically induced cell cycle arrest is caused by inactivation of enzymes responsible for nucleotide synthesis, ultimately inhibiting DNA replication (19, 39). However, inhibition of nucleotide synthesis occurs only under severe hypoxia (0.01% oxygen) or anoxia, but not under moderate hypoxia (0.1 to ϳ1% oxygen) (10).In the moderately hypoxic microenvironment, various biological reactions show significant changes relative to normoxia. Numerous studies on moderate hypoxia have indicated tha...