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Kyoji Ikeda

Publications and source records attributed to Kyoji Ikeda.

31 records · Page 2Linked to original sources
PubMed2003

Gamma-glutamyltranspeptidase stimulates receptor activator of nuclear factor-kappaB ligand expression independent of its enzymatic activity and serves as a pathological bone-resorbing factor.

A novel bone-resorbing factor was cloned using an expression cloning technique, which involved a Xenopus oocyte expression system and an assay for osteoclast formation. A candidate clone was isolated from a BW5147 mouse T-lymphoma cell cDNA library. Sequencing analysis identified the factor as gamma-glutamyltranspeptidase (GGT), which is an enzyme involved in glutathione metabolism. The addition of purified GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. An antibody against GGT inhibited osteoclast formation but not the enzymatic activity. We also demonstrated that an inactive form of GGT, the enzymatic activity of which had been blocked by chemical modification with a specific inhibitor, acivicin, supported osteoclast formation. These results indicate that GGT acts on osteoclast formation independent of its own enzymatic activity. Furthermore, both native GGT and inactive GGT stimulated the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein from bone marrow stromal cells. This report is the first demonstration of a novel biological activity of GGT protein in a manner independent of its enzymatic activity.

Animals↗
PubMed2003

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 G(1) 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 H(2)O(2) 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 beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-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.

Ataxia Telangiectasia↗
PubMed2003

A novel mitochondrial carnitine-acylcarnitine translocase induced by partial hepatectomy and fasting.

The carnitine-dependent transport of long-chain fatty acids is essential for fatty acid catabolism. In this system, the fatty acid moiety of acyl-CoA is transferred enzymatically to carnitine, and the resultant product, acylcarnitine, is imported into the mitochondrial matrix through a transporter named carnitine-acylcarnitine translocase (CACT). Here we report a novel mammalian protein homologous to CACT. The protein, designated as CACL (CACT-like), is localized to the mitochondria and has palmitoylcarnitine transporting activity. The tissue distribution of CACL is similar to that of CACT; both are expressed at a higher level in tissues using fatty acids as fuels, except in the brain, where only CACL is expressed. In addition, CACL is induced by partial hepatectomy or fasting. Thus, CACL may play an important role cooperatively with its homologue CACT in a stress-induced change of lipid metabolism, and may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.

3T3 Cells↗
PubMed2003

Loss of p27(Kip1) accelerates DNA replication after partial hepatectomy in mice.

BACKGROUND: Liver regeneration after partial hepatectomy (PH) is accomplished by a synchronous replication of hepatocytes. Both positive and negative regulators of cyclin-dependent protein kinase (Cdk) have been implicated in hepatocyte proliferation, but their specific roles in vivo remain to be clarified. To investigate the specific role of p27(Kip1), a member of the Cip/Kip family of Cdk inhibitors, in cell-cycle regulation during liver regeneration, p27-knockout mice were studied after PH. MATERIALS AND METHODS: Under ether anesthesia, mice were subjected to 70% PH. Animals were sacrificed at intervals after the surgery, and the remnant liver was harvested and analyzed. RESULTS: In p27-deficient mice, the timing of DNA synthesis was significantly accelerated with a perturbation in the ordered distribution of proliferating cells in the hepatic lobule. p27 deficiency, however, did not affect the whole population of cycling cells, the number of apoptotic cells, or liver injury and mortality after PH. CONCLUSION: These data provide in vivo evidence that p27 functions as a brake in the "start" of the hepatocyte cell cycle, thereby coordinating temporally and spatially the onset of DNA synthesis of hepatocytes within the hepatic lobules.

Animals↗
PubMed2002

Recruitment of NBS1 into PML oncogenic domains via interaction with SP100 protein.

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, chromosomal instability, radiation sensitivity, and an increased incidence of malignancies. NBS1, the protein responsible for NBS, forms a complex with MRE11 and RAD50, and plays a vital role in DNA repair, cell cycle checkpoint, and telomere maintenance. Here, we show that a BRCA carboxyl terminus (BRCT) domain-containing region of NBS1 interacts with a nuclear dots-associated protein, SP100. The SP100 and NBS1 proteins co-localized in PODs and APBs in normal human fibroblast MRC5 and ALT line VA13 at G2 phase, respectively. Introduction of PML and SP100 into NT2 cells, which express no detectable amount of PML or SP100 proteins, resulted in localization of NBS1 in ectopically expressed PODs. These results indicate that NBS1 is recruited into PODs via interaction with SP100 protein. Thus, interaction between the NBS1 and SP100 proteins may be involved in genomic stability and telomere maintenance.

Animals↗
PubMed2002

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 G(1)/S cell cycle checkpoint, but not the G(2)/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.

Animals↗
PubMed2002

FOXO forkhead transcription factors induce G(2)-M checkpoint in response to oxidative stress.

Members of the FOXO family of mammalian forkhead transcription factors, including AFX, FKHRL1, and FKHR, are homologs of DAF-16, which regulates genes that contribute both to longevity and to resistance to various stresses (including oxidative stress) in Caenorhabditis elegans. We have generated mouse myoblastic C2C12 cell lines in which expression of a constitutively active form of AFX (AFX-TM) is inducible by Cre-mediated recombination at loxP sites. Here we show that forced expression of AFX-TM blocked cell cycle progression at the G(1) and G(2) phases and that FOXO family members regulated the expression of stress-inducible genes such as GADD45. AFX and FKHRL1 each directly activated the GADD45 promoter through interaction with FOXO binding motifs. Oxidative stress activated the GADD45 promoter in a FOXO-dependent manner, resulting in an increased abundance of GADD45 mRNA and protein as well as G(2) arrest. These responses were evident in cells in which the tumor suppressor protein p53 was inactivated. Our results suggest that the FOXO family of transcription factors plays an important role in the regulation of GADD45 in response to oxidative stress and thereby contributes to G(2)-M checkpoint.

Adenoviridae↗
PubMed2002

A RING finger protein Praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Necdin family protein, Dlxin-1.

Msx2 and Dlx5 are homeodomain proteins that play an important role in osteoblast differentiation and whose expression is induced by bone morphogenetic proteins. Recently we have identified a novel protein, Dlxin-1, that associates with these homeodomain proteins and regulates Dlx5-dependent transcriptional function (Masuda, Y., Sasaki, A., Shibuya, H., Ueno, N., Ikeda, K., and Watanabe, K. (2001) J. Biol. Chem. 276, 5331-5338). In an attempt to elucidate the molecular function of Dlxin-1, two closely related RING finger proteins, Praja1 and Neurodap-1, were isolated by yeast two-hybrid screening using the C-terminal necdin homology domain of Dlxin-1 as bait. Glutathione S-transferase pull-down and immunoprecipitation/Western blotting assays following co-transfection of Dlxin-1 and Praja1 revealed that Praja1 binds to the C-terminal necdin homology domain of Dlxin-1 in vitro and in vivo, respectively. Overexpression of Praja1 caused a decrease in Dlxin-1 protein level, which was reversed when a proteasome inhibitor was added. Overexpression of Praja1 with a mutation in the RING finger inhibited the decrease in Dlxin-1 protein, pointing to the importance of ubiquitin-protein isopeptide ligase (E3) activity associated with RING finger. Wild-type Praja1, but not its RING finger mutant, promoted ubiquitination of Dlxin-1 in vivo. Finally, expression of Praja1 down-regulated Dlx5-dependent transcriptional activity in a GAL4-dependent assay. These results suggest that Praja1 regulates the transcription function of the homeodomain protein Dlx5 by controlling the stability of Dlxin-1 via an ubiquitin-dependent degradation pathway.

Antigens, Neoplasm↗
PubMed2002

Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambda-deficient mice: possible implication for the pathogenesis of immotile cilia syndrome.

A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol beta within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) 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 lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) 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 lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.

Animals↗
PubMed2002

Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow.

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1alpha,25-dihydroxyvitamin D3 [1a,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1alpha,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D-treated mice as well as estrogen-treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the management of osteoporosis.

Animals↗
PubMed2002

Role of inducible nitric oxide synthase in skeletal adaptation to acute increases in mechanical loading.

To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we examined inducible NO synthase (iNOS) gene knockout mice in the tail-suspension model. Histomorphometric analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes. Both iNOS+/+ and iNOS+/- mice responded to subsequent 14-day reloading, with increases in BV/TV and BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS-/- mice. The osteoblasts flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after reloading were rescued in iNOS-/- mice by treatment with an NO donor nitroglycerine (NG). Conversely, the responses in iNOS+/+ mice were inhibited by treatment with an NOS inhibitor aminoguanidine (AG). In bone marrow cell cultures, mineralized nodules derived from iNOS-/- mice after reloading were significantly reduced. Taken together, our results suggest that NO generated by iNOS in osteoblasts plays a critical role in adjusting bone turnover and increasing osteogenic activity in response to the acute increase in mechanical loading after tail suspension.

Adaptation, Physiological↗
PubMed2002

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Journal Article↗