Atsushi Hirao

Tie2/Angiopoietin-1 Signaling Regulates Hematopoietic Stem Cell Quiescence in the Bone Marrow Niche

The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.

Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells

The ‘ataxia telangiectasia mutated’ (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia–telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16INK4a-retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress.

Foxo3a Is Essential for Maintenance of the Hematopoietic Stem Cell Pool

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

Regulation of the HIF-1α Level Is Essential for Hematopoietic Stem Cells

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.

TGF-β–FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a+/+ and Foxo3a-/- mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-β is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-β inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-β inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-β–FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.

Chk2 Is a Tumor Suppressor That Regulates Apoptosis in both an Ataxia Telangiectasia Mutated (ATM)-Dependent and an ATM-Independent Manner

ABSTRACT In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM−/− and p53−/− mice, Chk2−/− mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2−/− mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G1/S arrest. Quantitative comparison of the G1/S checkpoint, apoptosis, and expression of p53 proteins in Chk2−/− versus ATM−/− thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2−/− thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3 + related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.

ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53.

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM−/− fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEFp53−/−. However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21CIP1 induction, resulting in partial release of the G2/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.

Activated macrophages promote Wnt signalling through tumour necrosis factor-α in gastric tumour cells

The activation of Wnt/β‐catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/β‐catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/β‐catenin activity in gastric tumorigenesis. We found β‐catenin nuclear accumulation in macrophage‐infiltrated dysplastic mucosa of the K19‐Wnt1 mouse stomach. Moreover, macrophage depletion in ApcΔ716 mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/β‐catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/β‐catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)‐α. Furthermore, treatment with TNF‐α induced glycogen synthase kinase 3β (GSK3β) phosphorylation, which resulted in the stabilization of β‐catenin. We also found that Helicobacter infection in the K19‐Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear β‐catenin accumulation. These results suggest that macrophage‐derived TNF‐α promotes Wnt/β‐catenin signalling through inhibition of GSK3β, which may contribute to tumour development in the gastric mucosa.

Regulation of Reactive Oxygen Species and Genomic Stability in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are defined by their ability both to self-renew and to give rise to fresh blood cells throughout the lifetime of an animal. The failure of HSCs to self-renew during aging is believed to depend on several intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) factors. In this review, we focus on how dysregulation of reactive oxygen species (ROS) and disruptions of genomic stability can impair HSC functions. Recently, it was shown that long-term self-renewing HSCs normally possess low levels of intracellular ROS. However, when intracellular ROS levels become excessive, they cause senescence or apoptosis, resulting in a failure of HSC self-renewal. Repression of intracellular ROS levels in HSCs by treatment with an antioxidant that scavenges ROS can rescue HSC functions, indicating that excess ROS levels are at the root of HSC failure. Products of numerous genes that are involved in either DNA-damage responses or longevity-related signaling contribute to the maintenance of the HSC self-renewal capacity. Further investigations on the molecular mechanisms of ROS regulation and on the manipulation of excess ROS levels could lead to the development of novel therapeutics for hematopoietic diseases, regenerative medicine, and the prevention of leukemia.

PI3K-Akt-mTORC1-S6K1/2 Axis Controls Th17 Differentiation by Regulating Gfi1 Expression and Nuclear Translocation of RORγ

The PI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation of CD4(+) T cells upon stimulation through TCR and CD28. Here, we demonstrate that the suppression of this axis by deletion of p85α or PI3K/mTORC1 inhibitors as well as T cell-specific deletion of raptor, an essential component of mTORC1, impairs Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of PI3K-Akt-mTORC1-S6K1 axis impairs the downregulation of Gfi1, a negative regulator of Th17 differentiation. Furthermore, we demonstrate that S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis, binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.