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Fuyuki Ishikawa

Publications and source records attributed to Fuyuki Ishikawa.

31 records · Page 2Linked to original sources
PubMed2003

Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression.

The Hairy-related bHLH proteins function as transcriptional repressors in most cases and play important roles in diverse aspects of metazoan development. Recently, it was shown that the Drosophila bHLH repressor proteins, Hairy and Deadpan, bind to and function with the NAD(+)-dependent histone deacetylase, Sir2. Here we demonstrate that the human Sir2 homologue, SIRT1, also physically associates with the human bHLH repressor proteins, hHES1 and hHEY2, both in vitro and in vivo. Moreover, using the reporter assay, we show that both SIRT1-dependent and -independent deacetylase pathways are involved in the transcriptional repressions mediated by these bHLH repressors. These results indicate that the molecular association between bHLH proteins and Sir2-related proteins is conserved among metazoans, from Drosophila to human, and suggest that the Sir2-bHLH interaction also plays important roles in human cells.

Animals↗
PubMed2003

Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

BACKGROUND: Cellular senescence is a state of irreversible growth arrest shown by normal cells, and has been most extensively studied in replicative senescence caused by telomere shortening. Several conditions, including oncogenic Ras over-expression and inappropriate culture conditions, also induce senescence without telomere shortening. However, it remains unclear how a common set of senescence phenotypes is indistinguishably induced in various types of senescence. RESULTS: We demonstrate that p38 mitogen-activated protein kinase (MAPK) plays important causative roles in senescent cells following telomere shortening, Ras-Raf activation, oxidative stress or inappropriate culture conditions. By monitoring the kinetics of p38 activation, we suggest that p38 is activated not directly by the initial stimuli, but in response to unidentified cellular conditions caused by these stimuli. Importantly, this p38-activating condition appears to be defined quantitatively as a sum of continuous and low-level stresses, and remains even after the initial stimuli are withdrawn, which may explain the well-known irreversible nature of cellular senescence. We also show that papilloma virus E7 abolishes the p38-induced growth arrest but not other senescence-associated phenotypes, indicating the differential role of pRb in the downstream of p38. CONCLUSION: These results indicate that p38 comprises the senescence-executing pathway in response to diverse stimuli.

Animals↗
PubMed2003

Cellular senescence, an unpopular yet trustworthy tumor suppressor mechanism.

The term "cellular senescence" refers to the state in which normal cells irreversibly stop dividing. Historically, this condition was first inferred from the finding that normal human fibroblasts cease dividing after a limited number of cell divisions. Since then, cellular senescence has been discussed as a potential cause of aging of organisms. However, recent studies have significantly expanded our view of cellular senescence in terms of both mechanistics and biological significance. Accordingly, cellular senescence is now considered to play an important adaptive role, namely, a tumor suppressor function. This review will focus on recent findings that have contributed to the elucidation of the adaptive role of cellular senescence.

Animals↗
PubMed2002

Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast.

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast, pku70(+), the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80(+), the gene encoding Ku80. Both pku70(+) and pku80(+) are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.

Antigens, Nuclear↗
PubMed2002

Telomerase rescues the expression levels of keratinocyte growth factor and insulin-like growth factor-II in senescent human fibroblasts.

Changes in expression levels of various cytokines, growth factors, and related genes were examined by reverse transcriptase polymerase chain reaction in a normal human fibroblast cell strain, TIG-3, along with in vitro aging. The expression levels of KGF and IGF-II were decreased with proliferative aging but not by growth arrest of young cells. In telomere-elongated cells prepared by transfection with human telomerase reverse transcriptase cDNA, high expression levels of these two genes were maintained, suggesting a causal relation between telomere shortening and reduced expression of KGF and IGF-II. The expression level of HGF was high in both growing and growth-arrested young cells but low in both senescent and telomere-elongated cells. The expression levels of follistatin and HB-EGF were high in both young growing and telomere-elongated cells but low in both senescent and growth-arrested young cells, indicating a growth-dependent expression. Expression levels of FGF-1, FGF-2, VEGF, BMP-3, and amphiregulin did not change with proliferative aging, growth arrest of young cells, or telomere elongation and life-span extension.

Aging↗
PubMed2002

MBD3 and HDAC1, two components of the NuRD complex, are localized at Aurora-A-positive centrosomes in M phase.

MBD3, a component of the histone deacetylase NuRD complex, contains the methyl-CpG-binding domain (MBD), yet does not possess appreciable mCpG-specific binding activity. The functional significance of MBD3 in the NuRD complex remains enigmatic, partly because of the limited availability of biochemical approaches, such as immunoprecipitation, to analyze MBD3. In this study, we stably expressed the FLAG-tagged version of MBD3 in HeLa cells. We found that MBD3-FLAG was incorporated into the NuRD complex, and the MBD3-FLAG-containing NuRD complex was efficiently immunoprecipitated by anti-FLAG antibodies. By exploiting this system, we found that MBD3 is phosphorylated in vivo in the late G(2) and early M phases. Moreover, we found that Aurora-A, a serine/threonine kinase active specifically in the late G(2) and early M phases, phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase. Interestingly, HDAC1 is distributed at the centrosomes in a manner similar to MBD3. These results suggest the highly dynamic nature of the temporal and spatial distributions, as well as the biochemical modification, of the NuRD complex in M phase, probably through an interaction with kinases, including Aurora-A. These observations will contribute significantly to the elucidation of the yet-uncharacterized cell cycle-controlled functions of the NuRD complex.

Aurora Kinases↗
PubMed2002

Successful inactivation of endogenous Oct-3/4 and c-mos genes in mouse preimplantation embryos and oocytes using short interfering RNAs.

Understanding oocyte maturation and early development in mammals is very important, especially because these cells serve as a source of materials useful in medical applications, such as ES cells. However, the limited availability of oocytes and embryos hampers the molecular dissection of the very early stage of mammalian development. Recently, the RNA interference technology has been acknowledged to be very effective and useful in diverse groups of cells, including mammalian cells. In this study, we examined whether short interfering RNAs (siRNAs) are applicable to mouse oocytes and preimplantation embryos, by targeting two genes, namely, Oct-3/4 and c-mos. siRNA injections successfully extinguished the production of these target genes. Moreover, the siRNA-injected oocytes and embryos showed phenotypes very similar to those exhibited by Oct-3/4- or Mos-knockout mice in previous studies. Accordingly, we concluded that siRNA is a useful tool in molecular studies on the early development of mouse.

Alkaline Phosphatase↗
PubMed2002

Cohesin defects lead to premature sister chromatid separation, kinetochore dysfunction, and spindle-assembly checkpoint activation.

Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules.

Cell Cycle Proteins↗
PubMed2002

The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.

Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.

Amino Acid Sequence↗
PubMed2002

Variant forms of upstream stimulatory factors (USFs) control the promoter activity of hTERT, the human gene encoding the catalytic subunit of telomerase.

It is known that Myc regulates the expression of TERT, the telomerase catalytic subunit gene, by binding to E box. Here we show that another E box-binding protein, upstream stimulatory factor (USF), also regulates TERT expression. Specifically, the N-terminally truncated form of USF2 is present in telomerase-negative/resting human lymphocytes, but not in telomerase-positive/phytohemagglutinin-activated lymphocytes. In electrophoretic mobility shift assay, both full-length and truncated USF2s bound to the TERT E box. In a transient expression assay, the truncated USF had a dominant-negative effect on both exogenous full-length USF and endogenous positive regulators for activating TERT expression. These results suggest that the differential abundance of truncated USF2 may regulate telomerase activity during lymphocyte activation.

Binding Sites↗
PubMed2002

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice.

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

Animals↗
PubMed2002

Stretch PCR assay.

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Autoradiography↗