Noboru Motoyama

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.

DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2–M checkpoint defect close to that observed in ATM−/− cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

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-deficient mice exhibit radioresistance and defective p53-mediated transcription

The mammalian Chk2 kinase is thought to mediate ATM‐dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2‐deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR‐induced apoptosis. The IR‐induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR‐induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild‐type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR‐dependent but Chk2‐independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53‐dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.

NAK is an IκB kinase-activating kinase

Phosphorylation of IκB by the IκB kinase (IKK) complex is a critical step leading to IκB degradation and activation of transcription factor NF-κB. The IKK complex contains two catalytic subunits, IKKα and IKKβ, the latter being indispensable for NF-κB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKβ activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-κB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IκB degradation and NF-κB activity through IKKβ. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-κB by protein kinase C-ε (PKCε). Thus, NAK is an IKK kinase that may mediate IKK and NF-κB activation in response to growth factors that stimulate PKCε activity.

NAK is an IkappaB kinase-activating kinase.

Phosphorylation of IκB by the IκB kinase (IKK) complex is a critical step leading to IκB degradation and activation of transcription factor NF-κB. The IKK complex contains two catalytic subunits, IKKα and IKKβ, the latter being indispensable for NF-κB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKβ activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-κB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IκB degradation and NF-κB activity through IKKβ. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-κB by protein kinase C-ε (PKCε). Thus, NAK is an IKK kinase that may mediate IKK and NF-κB activation in response to growth factors that stimulate PKCε activity.

Hydrocephalus, Situs Inversus, Chronic Sinusitis, and Male Infertility in DNA Polymerase λ-Deficient Mice: Possible Implication for the Pathogenesis of Immotile Cilia Syndrome

ABSTRACT A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase λ (Pol λ; also known as Pol β2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol β within the polymerase domain. With the use of homologous recombination, we generated Pol λ−/− mice. Pol λ−/− mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol λ−/− mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol λ−/− mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol λ−/− mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol λ−/− cells to various kinds of DNA damage is indistinguishable from that of Pol λ+/+ cells. Collectively, Pol λ−/− mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.

ATM–Chk2–p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency

BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full‐length Brca1 (Brca1Δ11/Δ11) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1Δ11/Δ11p53+/− mice display cancer susceptibility and premature aging. Here, we show that reduced expression and/or the absence of Chk2 allow Brca1Δ11/Δ11 mice to escape from embryonic lethality. Compared to Brca1Δ11/Δ11p53+/− mice, lifespan of Brca1Δ11/Δ11Chk2−/− mice was remarkably extended. Analysis of Brca1Δ11/Δ11Chk2−/− mice revealed that p53‐dependent apoptosis and growth defect caused by Brca1 deficiency are significantly attenuated in rapidly proliferating organs. However, in later life, Brca1Δ11/Δ11Chk2−/− female mice developed multiple tumors. Furthermore, haploid loss of ATM also rescued Brca1 deficiency‐associated embryonic lethality and premature aging. Thus, in response to Brca1 deficiency, the activation of the ATM–Chk2–p53 signaling pathway contributes to the suppression of neoplastic transformation, while leading to compromised organismal homeostasis. Our data highlight how accurate maintenance of genomic integrity is critical for the suppression of both aging and malignancy, and provide a further link between aging and cancer.

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts.

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G1 checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H2O2 in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated β-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21WAF1, and p16INK4A. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H2O2-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.

Necessary and Sufficient Role for a Mitosis Skip in Senescence Induction

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.