研究者総覧

冨永 薫 (トミナガ カオル)

  • 生化学講座(機能生化学部門) 教授
メールアドレス: tominagajichi.ac.jp
Last Updated :2021/12/04

研究者情報

学位

  • 博士(医学)(1994年03月 自治医科大学)

ホームページURL

J-Global ID

研究キーワード

  • ミトコンドリア   老化   代謝   エピジェネティクス   ガン   加齢   ヒストン修飾   幹細胞   クロマチン   細胞老化   リポ多糖   C3H   MAPキナーゼ   IL-6   マクロファージ   HeJマウス   線維芽細胞   NF-κB   感染防御   スカベンジャー受容体   LPS(リポ多糖)   ノックアウトマウス   

研究分野

  • ライフサイエンス / ゲノム生物学
  • ライフサイエンス / 実験動物学
  • ライフサイエンス / 腫瘍生物学
  • ライフサイエンス / 細胞生物学
  • ライフサイエンス / 医化学

経歴

  • 2020年10月 - 現在  自治医科大学医学部生化学講座機能生化学部門教授
  • 2011年04月  自治医科大学医学部准教授

研究活動情報

論文

  • Rintaro Kuroda, Kaoru Tominaga, Katsumi Kasashima, Kenji Kuroiwa, Eiji Sakashita, Hiroko Hayakawa, Tom Kouki, Nobuhiko Ohno, Kensuke Kawai, Hitoshi Endo
    PLOS ONE 16 7 e0255355 - e0255355 2021年07月 
    Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.
  • Satsuki Miyata, Kaoru Tominaga, Eiji Sakashita, Masashi Urabe, Yoshiyuki Onuki, Akira Gomi, Takashi Yamaguchi, Makiko Mieno, Hiroaki Mizukami, Akihiro Kume, Keiya Ozawa, Eiju Watanabe, Kensuke Kawai, Hitoshi Endo
    Scientific reports 9 1 9787 - 9787 2019年07月 
    Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1R132H. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in β-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.
  • Naoki Iwamori, Kaoru Tominaga, Tetsuya Sato, Kevin Riehle, Tokuko Iwamori, Yasuyuki Ohkawa, Cristian Coarfa, Etsuro Ono, Martin M Matzuk
    Proceedings of the National Academy of Sciences of the United States of America 113 37 E5408-15  2016年09月 
    Splicing can be epigenetically regulated and involved in cellular differentiation in somatic cells, but the interplay of epigenetic factors and the splicing machinery during spermatogenesis remains unclear. To study these interactions in vivo, we generated a germline deletion of MORF-related gene on chromosome 15 (MRG15), a multifunctional chromatin organizer that binds to methylated histone H3 lysine 36 (H3K36) in introns of transcriptionally active genes and has been implicated in regulation of histone acetylation, homology-directed DNA repair, and alternative splicing in somatic cells. Conditional KO (cKO) males lacking MRG15 in the germline are sterile secondary to spermatogenic arrest at the round spermatid stage. There were no significant alterations in meiotic division and histone acetylation. Specific mRNA sequences disappeared from 66 germ cell-expressed genes in the absence of MRG15, and specific intronic sequences were retained in mRNAs of 4 genes in the MRG15 cKO testes. In particular, introns were retained in mRNAs encoding the transition proteins that replace histones during sperm chromatin condensation. In round spermatids, MRG15 colocalizes with splicing factors PTBP1 and PTBP2 at H3K36me3 sites between the exons and single intron of transition nuclear protein 2 (Tnp2). Thus, our results reveal that MRG15 is essential for pre-mRNA splicing during spermatogenesis and that epigenetic regulation of pre-mRNA splicing by histone modification could be useful to understand not only spermatogenesis but also, epigenetic disorders underlying male infertile patients.
  • Mashiko T, Sakashita E, Kasashima K, Tominaga K, Kuroiwa K, Nozaki Y, Matsuura T, Hamamoto T, Endo H
    The Journal of biological chemistry 291 29 14996 - 15007 2016年07月 [査読有り][通常論文]
     
    Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.
  • Kaoru Tominaga
    Pathobiology of aging & age related diseases 5 27743 - 27743 2015年 
    Cellular senescence is a state of permanent growth arrest and is thought to play a pivotal role in tumor suppression. Cellular senescence may play an important role in tumor suppression, wound healing, and protection against tissue fibrosis in physiological conditions in vivo. However, accumulating evidence that senescent cells may have harmful effects in vivo and may contribute to tissue remodeling, organismal aging, and many age-related diseases also exists. Cellular senescence can be induced by various intrinsic and extrinsic factors. Both p53/p21 and p16/RB pathways are important for irreversible growth arrest in senescent cells. Senescent cells secret numerous biologically active factors. This specific secretion phenotype by senescent cells may largely contribute to physiological and pathological consequences in organisms. Here I review the molecular basis of cell cycle arrest and the specific secretion phenotype in cellular senescence. I also summarize the current knowledge of the role of cellular senescence in vivo in physiological and pathological settings.
  • Satoshi Yamamoto, Yasumitsu Nagao, Kenji Kuroiwa, Yoji Hakamata, Masaru Ichida, Fumiko Saito-Ohara, Kaoru Tominaga, Hitoshi Endo
    TRANSGENIC RESEARCH 23 5 757 - 765 2014年10月 [査読有り][通常論文]
     
    We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.
  • Syuichi Tetsuka, Kaoru Tominaga, Eriko Ohta, Kenji Kuroiwa, Eiji Sakashita, Katsumi Kasashima, Toshiro Hamamoto, Michito Namekawa, Mitsuya Morita, Shinsuke Natsui, Tatsuo Morita, Keiko Tanaka, Yoshihisa Takiyama, Imaharu Nakano, Hitoshi Endo
    JOURNAL OF THE NEUROLOGICAL SCIENCES 335 1-2 48 - 57 2013年12月 [査読有り][通常論文]
     
    Onconeural immunity, a cancer-stimulated immune reaction that cross-reacts with neural tissues, is considered to be the principal pathological mechanism for paraneoplastic neurological syndromes (PNS). A common PNS is paraneoplastic cerebellar degeneration (PCD). We had encountered a PCD patient with urothelial carcinomas (UC) of the urinary bladder who was negative for the well-characterized PNS-related onconeural antibodies. In the present study, we aimed to identify a new PCD-related onconeural antibody, capable of recognizing both cerebellar neurons and cancer tissues from the patient, and applied a proteomic approach using mass spectrometry. We identified anti-creatine kinase, brain-type (CKB) antibody as a new autoantibody in the serum and cerebrospinal fluid from the patient. Immunohistochemistry indicated that anti-CKB antibody reacted with both cerebellar neurons and UC of the urinary bladder tissues. However, anti-CKB antibody was not detected in sera from over 30 donors, including bladder cancer patients without PCD, indicating that anti-CKB antibody is required for onset of PCD. We also detected anti-CKB antibody in sera from three other PCD patients. Our study demonstrated that anti-CKB antibody may be added to the list of PCD-related autoantibodies and may be useful for diagnosis of PCD. (C) 2013 Elsevier B.V. All rights reserved.
  • Kotaro Kawanishi, Mitsuya Morita, Keiichi Nakahara, Syuichi Tetsuka, Tom Kouki, Kaoru Tominaga, Hitoshi Endo, Takashi Yashiro, Keiko Tanaka, Imaharu Nakano
    Brain and Nerve 65 11 1401 - 1405 2013年11月 [査読有り][通常論文]
     
    A 66-year-old man was diagnosed with bladder cancer at our urology department. Three months later, he developed subacute progressive cerebellar limb ataxia and truncal oscillation. Analysis of cerebrospinal fluid showed pleocytosis and increased concentrations of protein, while brain magnetic resonance imaging revealed no abnormalities. Based on the presence of the bladder cancer, the etiology of subacute cerebellar ataxia could be a paraneoplastic neurological syndrome. Four months later, the patient underwent transurethral resection of the bladder tumor, which was identified as urothelial cancer on the basis of pathological examinations. However, this procedure failed to improve his neurological symptoms. Serum paraneoplastic markers such as anti-Yo, anti-Hu, anti-Tr, and other antibodies were not detected. Immunohistochemical staining of mouse cerebellum using the patient's serum revealed coarse granular staining in the cytoplasm of Purkinje cells and diffuse staining in the neuropil of the molecular layer, suggesting the presence of an unknown antibody. Subsequently, one-dimensional electrophoresis western blotting using the patient's serum revealed several bands including a strong positive band of approximately 45 kDa in mouse cerebellum lysates but not in liver lysates. These bands have never been detected in sera derived from healthy donors. These results suggested the presence of a novel antibody in the patient's serum that might recognize the approximately 45 kDa protein related to paraneoplastic cerebellar degeneration. Cases of paraneoplastic neurological syndrome associated with bladder cancer have rarely been reported. We concluded that the present case may be categorized as paraneoplastic neurological syndrome caused by an unknown antibody.
  • Akimoto C, Sakashita E, Kasashima K, Kuroiwa K, Tominaga K, Hamamoto T, Endo H
    Biochimica et biophysica acta 1830 3 2728 - 2738 3 2013年03月 [査読有り][通常論文]
     
    BACKGROUND: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. METHODS: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. RESULTS: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts. CONCLUSIONS: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. GENERAL SIGNIFICANCE: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.
  • Chizuru Akimoto, Eiji Sakashita, Katsumi Kasashima, Kenji Kuroiwa, Kaoru Tominaga, Toshiro Hamamoto, Hitoshi Endo
    Biochimica et Biophysica Acta - General Subjects 1830 3 2728 - 2738 2013年03月 [査読有り][通常論文]
     
    Background Upstream open reading frames (uORFs) are commonly found in the 5′-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. Methods Northern blotting, 3′-RACE, and bioinformatics were used for determining the length of transcripts and their 3′ ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. Results The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5′-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5′-UTR are atypical but surely exist in human transcripts. Conclusions Multiple uORFs at the 5′-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. General significance Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs. © 2012 Elsevier B.V. ALl Rights Reserved.
  • Kaoru Tominaga, Olivia M. Pereira-Smith
    CURRENT DRUG TARGETS 13 13 1593 - 1602 2012年12月 [査読有り][通常論文]
     
    Cellular senescence is a state of irreversible growth arrest and thought to be a tumor suppressive mechanism. In addition, it has been reported that cellular senescence may play an important role in wound healing, tissue remodeling, organismal aging and age-related diseases. This loss of ability to divide, associated with senescence, is induced by factors that are intrinsic, such as genetically defined pathways and telomere erosion, and extrinsic eg. DNA damage, oxidative stress, over-expression of oncogenes and inadequate growth conditions. The p53/p21 and RB/p16 pathways are key to the cell cycle arrest associated with cellular senescence. Extensive molecular changes occur when cells become senescent, as gene expression profiling of senescent versus young cells has demonstrated, and this is, in part, due to alterations in chromatin structure. Here, we review the molecular basis of the cell cycle arrest in cellular senescence, focusing on chromatin regulation. We also summarize our current knowledge of the role of cellular senescence in vivo.
  • Viviana I. Perez, Lisa A. Cortez, Christie M. Lew, Marisela Rodriguez, Celeste R. Webb, Holly Van Remmen, Asish Chaudhuri, Wenbo Qi, Shuko Lee, Alex Bokov, Wilson Fok, Dean Jones, Arlan Richardson, Junji Yodoi, Yiqiang Zhang, Kaoru Tominaga, Gene B. Hubbard, Yuji Ikeno
    JOURNALS OF GERONTOLOGY SERIES A-BIOLOGICAL SCIENCES AND MEDICAL SCIENCES 66 12 1286 - 1299 2011年12月 [査読有り][通常論文]
     
    We examined the effects of increased levels of thioredoxin 1 (Trx1) on resistance to oxidative stress and aging in transgenic mice overexpressing Trx1 [Tg(TRX1)(+/0)]. The Tg(TRX1)(+/0) mice showed significantly higher Trx1 protein levels in all the tissues examined compared with the wild-type littermates. Oxidative damage to proteins and levels of lipid peroxidation were significantly lower in the livers of Tg(TRX1)(+/0) mice compared with wild-type littermates. The survival study demonstrated that male Tg(TRX1)(+/0) mice significantly extended the earlier part of life span compared with wild-type littermates, but no significant life extension was observed in females. Neither male nor female Tg(TRX1)(+/0) mice showed changes in maximum life span. Our findings suggested that the increased levels of Trx1 in the Tg(TRX1)(+/0) mice were correlated to increased resistance to oxidative stress, which could be beneficial in the earlier part of life span but not the maximum life span in the C57BL/6 mice.
  • AndreAna N. Pena, Kaoru Tominaga, Olivia M. Pereira-Smith
    EXPERIMENTAL CELL RESEARCH 317 11 1534 - 1540 2011年07月 [査読有り][通常論文]
     
    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in He La cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription. (C) 2011 Elsevier Inc. All rights reserved.
  • Meizhen Chen, Olivia M. Pereira-Smith, Kaoru Tominaga
    STEM CELL RESEARCH 7 1 75 - 88 2011年07月 [査読有り][通常論文]
     
    Chromatin regulation is crucial for many biological processes such as transcriptional regulation, DNA replication, and DNA damage repair. We have found that it is also important for neural stem/progenitor cell (NSC) function and neurogenesis. Here, we demonstrate that expression of the cyclin-dependent kinase inhibitor p21 is specifically up-regulated in Mrg15 deficient NSCs. Knockdown of p21 expression by p21 shRNA results in restoration of cell proliferation. This indicates that p21 is directly involved in the growth defects observed in Mrg15 deficient NSCs. Activated p53 accumulates in Mrg15 deficient NSCs and this most likely accounts for the up-regulation of p21 expression in the cells. We observed decreased p53 and p21 levels and a concomitant increase in the percentage of BrdU positive cells in Mrg15 null cultures following expression of p53 shRNA. DNA damage foci, as indicated by immunostaining for gamma H2AX and 53BP1, are detectable in a sub-population of Mrg15 deficient NSC cultures under normal growing conditions and the majority of p21-positive cells are also positive for 53BP1 foci. Furthermore, Mrg15 deficient NSCs exhibit severe defects in DNA damage response following ionizing radiation. Our observations highlight the importance of chromatin regulation and DNA damage response in NSC function and maintenance. (C) 2011 Elsevier B.V. All rights reserved.
  • Martrat G, Maxwell CM, Tominaga E, Porta-de-la-Riva M, Bonifaci N, Gómez-Baldó L, Bogliolo M, Lázaro C, Blanco I, Brunet J, Aguilar H, Fernández-Rodríguez J, Seal S, Renwick A, Rahman N, Kühl J, Neveling K, Schindler D, Ramírez MJ, Castellà M, Hernández G, EMBRACE, Easton DF, Peock S, Cook M, Oliver CT, Frost D, Platte R, Evans DG, Lalloo F, Eeles R, Izatt L, Chu C, Davidson R, Ong KR, Cook J, Douglas F, Hodgson S, Brewer C, Morrison PJ, Porteous M, Peterlongo P, Manoukian S, Peissel B, Zaffaroni D, Roversi G, Barile M, Viel A, Pasini B, Ottini L, Putignano AL, Savarese A, Bernard L, Radice P, Healey S, Spurdle A, Chen X, Beesley J, kConFab, Rookus MA, Verhoef S, Tilanus-Linthorst MA, Vreeswijk MP, Asperen CJ, Bodmer D, Ausems MG, van Os TA, Blok MJ, Meijers-Heijboer HE, Hogervorst FB, HEBON, Goldgar DE, Buys S, John EM, Miron A, Southey M, Daly MB, BCFR, SWE-BRCA, Harbst K, Borg A, Rantala J, Barbany-Bustinza G, Ehrencrona H, Stenmark-Askmalm M, Kaufman B, Laitman Y, Milgrom R, Friedman E, Domchek SM, Nathanson KL, Rebbeck TR, Johannsson OT, Couch FJ, Wang X, Fredericksen Z, Cuadras D, Moreno V, Pientka FK, Depping R, Caldés T, Osorio A, Benítez J, Bueren J, Heikkinen T, Nevanlinna H, Hamann U, Torres D, Caligo MA, Godwin AK, Imyanitov EN, Janavicius R, GEMO Study Collaborators, Sinilnikova OM, Stoppa-Lyonnet D, Mazoyer S, Verny-Pierre C, Castera L, de Pauw A, Bignon YJ, Uhrhammer N, Peyrat JP, Vennin P, Ferrer SF, Collonge-Rame MA, Mortemousque I, McGuffog L, Chenevix-Trench G, Pereira-Smith OM, Antoniou AC, Cerón J, Tominaga K, Surrallés J, Pujana MA
    Breast cancer research : BCR 13 R40  2 2011年04月 [査読有り][通常論文]
  • Mahlke MA, Cortez LA, Ortiz MA, Rodriguez M, Uchida K, Shigenaga MK, Lee S, Zhang Y, Tominaga K, Hubbard GB, Ikeno Y
    Pathobiology of aging & age related diseases 1 2011年 [査読有り][通常論文]
  • Hongjun Zhang, Yishi Li, Junsheng Yang, Kaoru Tominaga, Olivia M. Pereira-Smith, John Tower
    EXPERIMENTAL GERONTOLOGY 45 11 825 - 833 2010年11月 [査読有り][通常論文]
     
    The mammalian MRG15 gene encodes a chromodomain protein predicted to bind to chromatin via methylated histone tails Human MORF4 encodes a related but truncated protein that is capable of promoting cellular senescence in a subset of human tumor cell lines Drosophila contains a single homolog of human MRG15 called DmMRG15 Null mutation of MRG15 is embryonic-lethal in mice and Drosophila making the study of MRG15 requirements in adults difficult In these studies the DmMRG15 gene was over-expressed in Drosophila during developmental stages and in adults using a doxycycline-regulated system (Tet on) In addition an inverted-repeated construct was designed to inactivate DmMRG15 via the RNAi pathway and RNAI constructs were expressed using both the Tet-on system and Geneswitch system The DmMRG15 protein was readily expressed in adult flies in a doxycycline dependent manner A truncated form of DmMRG15 (called DmMT1) was designed to mimic the structure of human MORF4 and expression of this mutant protein or the inverted-repeat constructs inhibited fertility in females Conditional expression of the DmMRG15 inverted-repeat constructs during larval development or in adults caused reductions in survival These experiments indicate that Drosophila DmMRG15 gene function is required for female fertility larval survival and adult life span and provide reagents that should be useful for further dissecting the role of DmMRG15 in cell proliferation and aging (C) 2010 Elsevier Inc All rights reserved
  • Reini F. Luco, Qun Pan, Kaoru Tominaga, Benjamin J. Blencowe, Olivia M. Pereira-Smith, Tom Misteli
    SCIENCE 327 5968 996 - 1000 2010年02月 [査読有り][通常論文]
     
    Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery.
  • Kaoru Tominaga, Emiko Tominaga, Michael J. Ausserlechner, Olivia M. Pereira-Smith
    EXPERIMENTAL CELL RESEARCH 316 1 92 - 102 2010年01月 [査読有り][通常論文]
     
    After undergoing several rounds of divisions normal human fibroblasts enter a terminally nondividing state referred to as cellular or replicative senescence. We cloned MORF4 (mortality factor on human chromosome 4), as a cellular senescence inducing gene that caused immortal cells assigned to complementation group B for indefinite division to stop dividing. To facilitate analyses of this gene, which is toxic to cells at low levels, we obtained stable clones of HeLa cells expressing a tetracycline-induced MORF4 construct that could be induced by doxycycline in a dose-dependent manner. MORF4 induction resulted in reduced colony formation after 14 days of culture, as previously observed. We determined that MORF4 protein was unstable and that addition of the proteasome inhibitor MG132 resulted in the accumulation of the protein. Following removal of MG132 the protein was rapidly degraded. Subcellular fractionation following MG132 treatment demonstrated that the protein accumulates primarily in the cytoplasm with some amounts present in the nucleus. It is therefore possible that MORF4 protein, which escapes degradation in the cytoplasm, is transported to the nucleus where it is functional. The results suggest that levels of MORF4 in cells must be tightly controlled and one mechanism involves stability of the protein. (C) 2009 Elsevier Inc. All rights reserved.
  • Meizhen Chen, Kaoru Tominaga, Olivia M. Pereira-Smith
    AGING, CANCER, AND AGE-RELATED DISEASES: COMMON MECHANISM? 1197 134 - 141 2010年 [査読有り][通常論文]
     
    Cellular senescence is the dominant phenotype over immortality. In our studies to identify senescence-related genes, we cloned Morf4, which induced senescence in a subset of tumor cells. Morf4 is a member of a family of seven genes, and Morf-related genes (Mrg) on chromosomes 15 (Mrg15) and X (MrgX) are also expressed. In contrast to MORF4, MRG15 and MRGX are positive regulators of cell division. All three proteins interact with histone deacetylases and acetyltransferases, suggesting that they function in regulation of chromatin dynamics. Mrg15 knockout mice are embryonic lethal, and mouse embryonic fibroblasts derived from Mrg15 null embryos proliferate poorly, enter senescence rapidly, and have impaired DNA repair compared to the wild type. Mrg15 null embryonic neural stem and progenitor cells also have a decreased capacity for proliferation and differentiation. Further studies are needed to determine the function of this gene family in various biological processes, including neural stem and progenitor cell aging.
  • Ahmed Sabbah, Te Hung Chang, Rosalinda Harnack, Victoria Frohlich, Kaoru Tominaga, Peter H. Dube, Yan Xiang, Santanu Bose
    NATURE IMMUNOLOGY 10 10 1073 - U49 2009年10月 [査読有り][通常論文]
     
    Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs) and RIG-like helicase (RLH) receptors, are involved in innate immune antiviral responses. Here we show that nucleotide-binding oligomerization domain 2 (Nod2) can also function as a cytoplasmic viral PRR by triggering activation of interferon-regulatory factor 3 (IRF3) and production of interferon-beta (IFN-beta). After recognition of a viral ssRNA genome, Nod2 used the adaptor protein MAVS to activate IRF3. Nod2-deficient mice failed to produce interferon efficiently and showed enhanced susceptibility to virus-induced pathogenesis. Thus, the function of Nod2 as a viral PRR highlights the important function of Nod2 in host antiviral defense mechanisms.
  • Meizhen Chen, Masumi Takano-Maruyama, Olivia M. Pereira-Smith, Gary O. Gaufo, Kaoru Tominaga
    JOURNAL OF NEUROSCIENCE RESEARCH 87 7 1522 - 1531 2009年05月 [査読有り][通常論文]
     
    Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells (NPCs). Chromatin regulation is a key step in self-renewal activity and fate decision of NPCs. However, the molecular mechanism or mechanisms of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of NPCs. Neuroepithelia from Mrg15-deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated NPCs from Mrg15-deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15-deficient embryo grew less efficiently than those from wild type. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incorporation revealed that Mrg15-deficient NPCs have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15-deficient NPCs most likely accounts for the thinner neuroepithelia in Mrg15-deficient embryonic brain. Moreover, we also demonstrate Mrg15-deficient NPCs are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development. (C) 2008 Wiley-Liss, Inc.
  • Sandra N. Garcia, Bhakti M. Kirtane, Andrej J. Podlutsky, Olivia M. Pereira-Smith, Kaoru Tominaga
    FEBS LETTERS 581 27 5275 - 5281 2007年11月 [査読有り][通常論文]
     
    MORF4-related gene on chromosome 15 (MRG15) is a core component of the NuA4/Tip60 histone acetyltransferase complex that modifies chromatin structure. We here demonstrate that Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit an impaired DNA-damage response post gamma irradiation, when compared to wild-type cells. Defects in DNA-repair and cell growth, and delayed recruitment of repair proteins to sites of damage were observed. Formation of phosphorylated H2AX and 53BP1 foci was delayed in Mrg15 mutant versus wild-type cells following irradiation. These data implicate a novel role for MRG15 in DNA-damage repair in mammalian cells. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • BR Bowman, CM Moure, BM Kirtane, RL Welschhans, K Tominaga, OM Pereira-Smith, FA Quiocho
    STRUCTURE 14 1 151 - 158 2006年01月 [査読有り][通常論文]
     
    The ubiquitous MRG/MORF family of proteins is involved in cell senescence, or the terminal loss of proliferative potential, a model for aging and tumor suppression at the cellular level. These proteins are defined by the similar to 20 kDa MRG domain that binds a plethora of transcriptional regulators and chromatin-remodeling factors, including the histone deacetylase transcriptional corepressor mSin3A and the novel nuclear protein PAM14, and they are also known components of the Tip60/NuA4 complex via interactions with the MRG binding protein (MRGBP). We present here the crystal structure of a prototypic MRG domain from human MRG15 whose core consists of two orthogonal helix hairpins. Despite the lack of sequence similarity, the core structure has surprisingly striking homology to a DNA-interacting domain of the tyrosine site-specific recombinases XerD, X integrase, and Cre. Site-directed mutagenesis studies based on the X-ray structure and bioinformatics identified key residues involved in the binding of PAM14 and MRGBP.
  • K Tominaga, MM Matzuk, OM Pereira-Smith
    MOLECULAR AND CELLULAR BIOLOGY 25 12 4873 - 4880 2005年06月 [査読有り][通常論文]
     
    MRGX is one of the members of MORF4/MRG family of transcriptional regulators, which are involved in cell growth regulation and cellular senescence. We have shown that MRGX and MRG15 associate with Rb in nucleoprotein complexes and regulate B-myb promoter activity. To elucidate the functions of MRGX and to explore its potential role in modulating cell growth in vivo, we have generated MrgX-deficient mice. Characterization of the expression pattern of mouse MrgX demonstrated it was ubiquitously expressed in all tissues of adult mice and also during embryogenesis and overlapped with its homolog Mrg15. MRGX and MRG15 proteins localize predominantly to the chromatin fraction in the nucleus, although a small amount of both proteins localized to the nuclear matrix. Whereas disruption of Mrg15 results in embryonic lethality, absence of MrgX did not impair mouse development and MrgX null mice are healthy and fertile. MrgX-deficient and wild-type mouse embryonic fibroblasts (MEFs) also had similar growth rates and showed no differences in cell cycle-related gene expression in response to serum stimulation. Mrg15 expression in MrgX-deficient tissues and MEFs was not upregulated compared with wild-type tissues and MEFs. MRG15 is highly conserved with orthologs present from humans to yeast and is essential for survival of mice. In contrast, MRGX, which evolved later, is expressed only in vertebrates, suggesting that the lack of phenotype of MrgX-deficient mice is secondary to a compensatory effect by the evolutionarily conserved MRG15 protein but not vice versa.
  • K Tominaga, B Kirtane, JG Jackson, Y Ikeno, T Ikeda, C Hawks, Smith, JR, MM Matzuk, OM Pereira-Smith
    MOLECULAR AND CELLULAR BIOLOGY 25 8 2924 - 2937 2005年04月 [査読有り][通常論文]
     
    MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15(-/-) embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15(-/-) mouse embryonic fibroblasts. The hearts of the Mrg15(-/-) embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15(-/-) embryos appeared pale, and microarray analysis revealed that a-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the a-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.
  • K Tominaga, DM Magee, MM Matzuk, OM Pereira-Smith
    MOLECULAR AND CELLULAR BIOLOGY 24 19 8366 - 8373 2004年10月 [査読有り][通常論文]
     
    PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).
  • K Tominaga, JK Leung, P Rookard, J Echigo, Smith, JR, OM Pereira-Smith
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 49 49618 - 49624 2003年12月 [査読有り][通常論文]
     
    MRGX is a novel transcription factor that is a member of the mortality factor 4 (MORF4)-related gene family. MRG15, a closely related family member, is in a complex with the retinoblastoma tumor suppressor protein Rb and activates the B-myb promoter, which is tightly controlled by Rb/E2F through the E2F binding site. In this study we investigated the effect of MRGX on the B-myb promoter. Interestingly, MRGX repressed the B-myb promoter in EJ cells ( human bladder carcinoma cells), which have a functional Rb, but activated B-myb in HeLa cells ( human cervical carcinoma cells), which express a lower amount of Rb. This repression and activation was dependent on the helix-loop-helix and leucine zipper regions of the MRGX protein but not the N-terminal region. MRGX interacts with Rb through the helix-loop-helix and leucine zipper regions. Using a treatment of trichostatin A, which is a potent inhibitor of histone deacetylase ( HDAC), we determined that the repression of the B-myb promoter by MRGX in EJ cells was dependent on HDAC activity. We confirmed the association of MRGX with HDAC1 by immunoprecipitation/ Western analysis and determined that MRGX complexes had HDAC activity. The data indicate that MRGX can repress or activate the B-myb promoter depending on the cell type studied, suggesting that there may be tissue-specific functions of this protein.
  • K Tominaga, OM Pereira-Smith
    GENE 294 1-2 215 - 224 2002年07月 [査読有り][通常論文]
     
    MORF4 (mortality factor on chromosome 4) and the novel related MRG (MORF4-related gene) gene family were identified when MORF4 was shown to induce senescence in a subset of tumor cell lines. The gene on chromosome 15 (MRG15) has high similarity to Drosophila MSL3, which is a component of the dosage compensation complex. MRG15 also has a chromodomain and may therefore function as a chromatin remodeling factor in a complex(es) involving a histone acetyltransferase, similar to MSL3. To complement our studies on human MRG15, we cloned and characterized the mouse MRG15 gene. Mouse MRG15 is expressed ubiquitously in adult tissues and at various embryonic stages, and expression in adult testis is higher than in other tissues. MRG15-b, which is an alternatively spliced form of MRG15-a and has a 39-amino-acid insertion in the chromodomain, is also expressed in all mouse tissues examined and localizes to the nucleus of cells. It is possible that MRG15-b may lack the function of the chromodomain because of the additional amino acids and could potentially be the equivalent of the human MORF4 in the mouse. The mouse MRG15 gene is composed of twelve exons and spans over 24 kb DNA. Using luciferase constructs we have determined that there is a functional promoter sequence 1.8 kb upstream of the ATG start codon. This region contains no TATA box but has GC-rich regions, consistent with the ubiquitous expression we have observed. (C) 2002 Elsevier Science B.V. All rights reserved.
  • K Tominaga, A Olgun, Smith, JR, OM Pereira-Smith
    MECHANISMS OF AGEING AND DEVELOPMENT 123 8 927 - 936 2002年04月 [査読有り][通常論文]
     
    Cellular senescence or replicative senescence is a state of irreversible growth arrest that somatic cells enter as a result of replicative exhaustion. This can be mimicked by culture manipulations such as Ras oncogene overexpression or treatment with various agents such as sodium butyrate and 5-azacytidine. It is believed that cellular senescence is one of the protective mechanisms against tumor formation. Genetic analyses of cellular senescence have revealed that it is dominant over immortality because whole cell fusion of normal with immortal cells yields hybrids with limited division potential. Only four complementation groups for indefinite division have been identified from extensive studies fusing different immortal human cell lines with each other. The senescence-related genes for three of the complementation groups B-D have been identified on human chromosomes 4, 1, and 7, respectively, by microcell-mediated chromosome transfer, though the existence of senescence-related genes on other chromosomes has been suggested. MORF4 was cloned as the senescence-related gene on human chromosome 4 and is a member of a new gene family, which has multiple transcription factor-like motifs. This gene family may affect cell division by modulating gene expression. Study of this novel gene family should lead to new insights regarding the mechanisms and function of cellular senescence in aging and immortalization. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
  • H Takai, K Tominaga, N Motoyama, YA Minamishima, H Nagahama, T Tsukiyama, K Ikeda, K Nakayama, N Nakanishi, K Nakayama
    GENES & DEVELOPMENT 14 12 1439 - 1447 2000年06月 [査読有り][通常論文]
     
    The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.
  • S Saito, M Matsuura, K Tominaga, T Kirikae, M Nakano
    EUROPEAN JOURNAL OF BIOCHEMISTRY 267 1 37 - 45 2000年01月 [査読有り][通常論文]
     
    The surface antigen CD14 is known to play a central role in the recognition of lipopolysaccharide by macrophages. We characterized a mutant cell line, J7.DEF.3, derived from a murine macrophage-like cell line, J774.1, to be defective in the ability to express the membrane-associated form of CD14 (mCD14) but not in the ability to release the soluble form of CD14 (sCD14), and used these parent and mutant cells to investigate the role of CD14 in lipopolysaccharide signaling. In response to lipopolysaccharide stimulation, mutant cells produced slightly less tumor necrosis factor than parent cells, and produced much less (negligible level) nitric oxide than parent cells. Production of both tumor necrosis factor and nitric oxide by parent cells upon lipopolysaccharide stimulation was suppressed by anti-CD14 serum. Expression of interferon-beta mRNA by stimulation with lipopolysaccharide, detected in parent cells, was barely detectable in mutant cells and in enzymatically mCD14-eliminated parent cells. Lipopolysaccharide-induced nitric oxide production in parent cells was suppressed by anti-(murine interferon-beta), and its production in the mutant cells appeared and increased dose dependently on exogenously supplied murine interferon-beta in the presence of lipopolysaccharide. These results provide new insight into the lipopolysaccharide signaling pathway, indicating that the lipopolysaccharide signal for interferon-beta production is transduced through a mCD14-dependent pathway and that the endogenously generated interferon-beta is an essential cofactor leading to nitric oxide production. Nuclear translocation of a transcription factor, nuclear factor kappa B, was observed in both parent and mutant cells following stimulation with a low dose of lipopolysaccharide, and mitogen-activated protein kinases were also activated in both types of cell, although a higher dose of lipopolysaccharide was required by the mutant cells than by the parent cells. These results indicate that these signaling factors may participate in the mCD14-independent lipopolysaccharide signaling pathway rather than in the mCD14-dependent interferon-beta-producing pathway.
  • K Tominaga, S Saito, M Matsuura, K Funatogawa, H Matsumura, M Nakano
    JOURNAL OF LEUKOCYTE BIOLOGY 66 6 974 - 980 1999年12月 [査読有り][通常論文]
     
    Murine peritoneal exudate cells (PEC) pre-exposed to bacterial lipopolysaccharide (LPS) show augmented nitric oxide (NO) production by LPS restimulation, in contrast to LPS tolerance with reduced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), Significant amounts of interferon-gamma (IFN-gamma) were detected in the PEC cultures on LPS stimulation, and anti-IFN-gamma antibody suppressed the LPS-induced NO, but not TNF-alpha and IL-6, production. Addition of anti-IFN-gamma antibody to the cultures in the LPS pre-exposure step strongly suppressed the augmented NO production on LPS restimulation. Anti-IL-12 antibody, which suppressed the LPS-induced IFN-gamma production, also suppressed the augmented NO production, as did anti-IFN-gamma antibody. Taken together, we propose the following mechanisms: (I) T and NK cells hr PEC produce IFN-gamma by the action of IL-12, which is derived from LPS-stimulated macrophages, and (2) the de novo-produced IFN-gamma activates macrophages to augment NO production on LPS restimulation.
  • K Tominaga, H Morisaki, Y Kaneko, A Fujimoto, T Tanaka, M Ohtsubo, M Hirai, H Okayama, K Ikeda, M Nakanishi
    JOURNAL OF BIOLOGICAL CHEMISTRY 274 44 31463 - 31467 1999年10月 [査読有り][通常論文]
     
    In response to DNA damage, mammalian cells adopt checkpoint regulation, by phosphorylation and stabilization of p53, to delay cell cycle progression. However, most cancer cells that lack functional p53 retain an unknown checkpoint mechanism(s) by which cells are arrested at the G(2)/M phase. Here we demonstrate that a human homolog of Cds1/Rad53 kinase (hCds1) is rapidly phosphorylated and activated in response to DNA damage not only in normal cells but in cancer cells lacking functional p53. A survey of various cancer cell lines revealed that the expression level of hCds1 mRNA is inversely related to the presence of functional p53. In addition, transfection of normal human fibroblasts with SV40 T antigen or human papilloma viruses E6 or E7 causes a marked induction of hCds1 mRNA, and the introduction of functional p53 into SV40 T antigen- and E6-, but not E7-, transfected cells decreases the hCds1 level, suggesting that p53 negatively regulates the expression of hCds1. In cells without functional ataxia telangiectasia mutated (ATM) protein, phosphorylation and activation of hCds1 were observed in response to DNA damage induced by UV but not by ionizing irradiation. These results suggest that hCds1 is activated through an ATM-dependent as well as -independent pathway and that it may complement the function of p53 in DNA damage checkpoints in mammalian cells.
  • Y Kaneko, N Watanabe, H Morisaki, H Akita, A Fujimoto, K Tominaga, M Terasawa, A Tachibana, K Ikeda, M Nakanishi
    ONCOGENE 18 25 3673 - 3681 1999年06月 [査読有り][通常論文]
     
    Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of CaS25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, we demonstrate that in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT) which lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM, Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216, which is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase.
  • K Tominaga, S Saito, M Matsuura, M Nakano
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1450 2 130 - 144 1999年06月 [査読有り][通常論文]
     
    Endotoxin/lipopolysaccharide (LPS) tolerance, a hyporesponsive state to endotoxin or LPS stimulation, was induced in murine peritoneal macrophages by previous exposure of macrophages to LPS. Expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNA in response to LPS stimulation was suppressed in LPS-tolerant macrophages. Tyrosine phosphorylations in response to LPS of 40-45-kDa proteins in non-tolerant macrophages were also suppressed in LPS-tolerant macrophages. These proteins corresponded to two members of the mitogen-activated protein kinase (MAPK) family, ERK and p38. In addition to these proteins, another MAPK family protein, JNK, was also suppressed in LPS-tolerant macrophages. Activation of Raf-1, located in the upstream portion of ERK cascades, was also suppressed by LPS-tolerance induction. These suppressions in LPS-tolerant macrophages were exhibited against stimulation by an LPS agonist like taxol, but not towards stimulation by an unrelated activator like phorbol ester (PMA). Activation of the transcription factor NF-kappa B, which is supposed to be one of the components of another important pathway for transduction of LPS-stimulated cytokine producing signals, was strongly suppressed and degradation of I kappa B, an inhibitor of NF-kappa B, was also severely diminished in LPS-tolerant macrophages. Although a monosaccharide lipid A analog, GLA-58, was able to stimulate macrophages to activate ERK proteins without cytokine production, pretreatment of macrophages with this compound suppressed both LPS-stimulated activation of ERK and cytokine production. Furthermore, downregulation of LPS-uptake in LPS-tolerant macrophages was not observed. Based on all these findings, LPS tolerance might be caused by the previous activation of some components on LPS-signaling pathways. This may then induce a refractory state in key LPS-signal transducer molecules located downstream of the cell membrane LPS receptor and upstream of the branching point in intracellular cascades for activation of MAPK and NF-kappa B, probably in some initial steps of intracellular signaling. (C) 1999 Elsevier Science B.V. All rights reserved.
  • M Nakano, K Tominaga, S Saito, F Kirikae, SN Lin, CL Fumero, Ojima, I, T Kirikae
    JOURNAL OF ENDOTOXIN RESEARCH 5 1-2 102 - 106 1999年 [査読有り][通常論文]
     
    LPS-tolerance, a state of refractoriness to LPS-stimulation, is induced in murine peritoneal macrophages by prior exposure to LPS. LPS-induced expression of TNF and IL-6 mRNA as well as activation of various intracellular kinases and factors, including ERK, p38, JNK, Raf-1 and NF-kappa B were all suppressed in LPS-tolerant macrophages; responses to stimulation by paclitaxel (Taxol(TM)), an LPS agonist, were similarly suppressed, but responses to phorbol esters (PMA) were unaffected. Binding and uptake of [I-125]-labeled LPS to tolerant macrophages was somewhat greater in tolerant than in non-tolerant macrophages. Thus, the refractory state appears to involve inhibition or blockade of LPS-signaling molecules located downstream of the cell membrane LPS receptor and upstream of the branch point in the intracellular cascades leading to activation of MAPK and NF-kappa B. LPS conditioning also suppressed LPS- and Taxol-induced TNF production, but augmented nitric oxide (NO) production. In contrast, Taxol conditioning failed to suppress LPS-induced TNF production. Conditioning with the synthetic taxoid analog, nor-seco-taxoid, which does not induce macrophage activation, enhanced LPS- and Taxol-induced NO production. These findings provide us with new information about the relationship between the LPS and Taxol receptors as well as about the signaling pathways leading to TNF and NO production.
  • M Tanikawa, K Yamada, K Tominaga, H Morisaki, Y Kaneko, K Ikeda, M Suzuki, T Kiho, K Tomokiyo, K Furuta, R Noyori, M Nakanishi
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 29 18522 - 18527 1998年07月 [査読有り][通常論文]
     
    Although the cyclopentenone prostaglandin A(1) (PGA(1)) is known to arrest the cell cycle at the G(1) phase in vitro and to suppress tumor growth in vivo, its relatively weak activity limits its usefulness in cancer chemotherapy, In an attempt to develop antitumor drugs of greater potency and conspicuous biological specificity, we synthesized novel analogs based on the structure of PGA(1). Of the newly synthesized analogs, 15-epi Delta(7)-PGA(1), methyl ester (NAG-0092), 12-iso-Delta(7)-PGA(1), methyl ester (NAG-0093), and ent-Delta(7)-PGA(1), methyl ester (NAG-0022) possess a cross conjugated dienone structure around the five-member ring with unnatural configurations at C(12) and/or C(15) and were found to be far more potent than native PGA(1) in inhibiting cell growth and causing G(1) arrest in A172 human glioma cells. These three analogs induced the expression of p21 at both RNA and protein levels in a time- and dose-dependent fashion. Kinase assays with A172 cells treated with these analogs revealed that both cyclin A- and E-dependent kinase activities were markedly reduced, although cyclin D1-dependent kinase activity was unaffected. Immunoprecipitation-Western blot analysis showed that the decrease in cyclin A-dependent kinase activity was due to an increased association of p21 with cyclin A-cyclin-dependent kinase 2 complexes, whereas the decrease in cyclin E-dependent activity was due to a combined mechanism involving reduction in cyclin E protein itself and increased association of p21. Thus, these newly synthesized PGA(1) analogs may prove to be powerful tools in cancer chemotherapy as well as in investigations of the structural basis of the antiproliferative activity of A series prostaglandins.
  • T Kirikae, F Kirikae, S Saito, K Tominaga, H Tamura, Y Uemura, T Yokochi, M Nakano
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 42 5 1015 - 1021 1998年05月 [査読有り][通常論文]
     
    The supernatants taken from Pseudomonas aeruginosa and Escherichia coli cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those taken from the same cultures in the presence of imipenem (IPM) yielded a very low level of endotoxin. The biological activities of endotoxin in the supernatants were compared with those of phenol water-extracted lipopolysaccharide (LPS), The endotoxin released from the organisms as a result of CAZ treatment (CAZ-released endotoxin) contained a large amount of protein. The protein, however, lacked endotoxic activity, since the endotoxin did not show any in vivo toxic effects in LPS-hyporesponsive C3H/HeJ mice sensitized with D-(+)-galactosamine (GalN) or any activation of C3H/HeJ mouse macrophages in vitro. The activities of CAZ- and IPM-released endotoxin (as assessed by a chromogenic Limulus test) were fundamentally the same as those of P. aeruginosa LPS, since their regression lines were parallel. The CAZ-released endotoxin was similar to purified LPS with respect to the following biological activities in LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice: lethal toxicity in GalN-sensitized mice, in vitro induction of tumor necrosis factor-and NO production by macrophages, and mitogen-activated protein kinase activation in macrophages. The macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on the presence of serum factor and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A).
  • Kikkawa, I, S Saito, K Tominaga, Y Hoshino, Y Ooi, M Nakano
    MICROBIOLOGY AND IMMUNOLOGY 42 9 591 - 598 1998年 [査読有り][通常論文]
     
    Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-alpha and nitric oxide (NO), Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-alpha and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-alpha production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-alpha and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-alpha and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells, The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N-G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA, Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA, These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.
  • K Tominaga, T Kirikae, M Nakano
    MOLECULAR IMMUNOLOGY 34 16-17 1147 - 1156 1997年11月 [査読有り][通常論文]
     
    Fibroblasts participate in inflammatory processes and non-specific immunity by producing cytokines and mediators in response to bacterial lipopolysaccharide (LPS). The detailed mechanism of LPS-induced cytokine production by fibroblasts has not been sufficiently studied. We isolated murine embryonic fibroblasts (MEF) from LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice and established MEF cell lines and MEF clones. Primarily cultured MEF, MEF cell lines and MEF clones from C3H/HeN mice (MEF.He) expressed interleukin (IL)-6 mRNA and produced IL-6 molecules in response to even a very low dose (1 ng/ml) of LPS. By contrast, those from C3H/HeJ mice (MEF.HeJ) neither expressed IL-6 mRNA nor produced IL-6 in response to 1 ng of LPS per mi, although they expressed IL-6 mRNA and produced IL-6 in response to high doses (more than 100 ng/ml) of LPS. The MEF.He clone, but not the MEF.HeJ clone, expressed IL-6 mRNA in response to taxol or ceramide, whereas MEF.HeJ clones as well as the MEF.He clone expressed IL-6 mRNA in response to IL-la. These results indicate that in the responses to LPS, taxol and ceramide, MEF retain the same reactivity as that of the mouse strains from which the MEF were derived, and LPS shares the IL-6 signal transduction pathway with taxol and ceramide, but not with IL-1. CD14 is not relevant to the LPS-induced IL-6 production by MEF, since cloned MEF.He and MEF.HeJ were shown not to express CD14 mRNA by Northern blot analysis. No difference in LPS-specific binding capacity was shown between the MEF.He and MEF.HeJ clones. This finding, together with the fact that hyporesponsiveness of MEF.HeJ to LPS was shown at the level of IL-6 mRNA expression, suggests that the defect in the LPS-induced IL-6 signal transduction pathway in MEF from C3H/HeJ mice is probably located at some site after the LPS-recognition site on the cell surface and before transcription of the IL-6 gene. (C) 1997 Elsevier Science Ltd. All rights reserved.
  • T Kirikae, F Kirikae, K Tominaga, N Qureshi, S Yamamoto, M Nakano
    JOURNAL OF ENDOTOXIN RESEARCH 4 2 115 - 122 1997年04月 [査読有り][通常論文]
     
    Paclitaxel (taxol), a microtubule stabilizer with anticancer activity, mimics the actions of lipopolysaccharide (LPS) on murine macrophages in vitro. Recent studies have shown that the Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) inhibits both LPS- and paclitaxel-induced activation of murine macrophages, and have suggested that LPS, RsDPLA, and paclitaxel share the same receptor site on murine macrophages. To analyze this receptor site, the present study focused on the interactions between LPS, RsDPLA and paclitaxel in the activation of ST2 cells derived from murine bone marrow stroma. The ST2 cells did not express CD14 mRNA. The cells produced IL-6 molecules and expressed IL-6 mRNA in response to LPS, but did not produce TNF and nitric oxide. Paclitaxel induced IL-6 mRNA expression in ST2 cells. RsDPLA inhibited both LPS- and paclitaxel-induced IL-6 mRNA expression in a dose-dependent manner. These results suggest that LPS, RsDPLA, and paclitaxel are recognized by the same receptor complex on ST2 cells, and that the receptor functions without membrane CD14.
  • T. Kirikae, F. Kirikae, K. Tominaga, N. Qureshi, S. Yamamoto, M. Nakano
    Innate Immunity 4 2 115 - 122 1997年 [査読有り][通常論文]
     
    Paclitaxel (taxol), a microtubule stabilizer with anticancer activity, mimics the actions of lipopolysaccharide (LPS) on murine macrophages in vitro. Recent studies have shown that the Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) inhibits both LPS- and paclitaxel-induced activation of murine macrophages, and have suggested that LPS, RsDPLA, and paclitaxel share the same receptor site on murine macrophages. To analyze this receptor site, the present study focused on the interactions between LPS, RsDPLA and paclitaxel in the activation of ST2 cells derived from murine bone marrow stroma. The ST2 cells did not express CD14 mRNA. The cells produced IL-6 molecules and expressed IL-6 mRNA in response to LPS, but did not produce TNF and nitric oxide. Paclitaxel induced IL-6 mRNA expression in ST2 cells. RsDPLA inhibited both LPS- and paclitaxel-induced IL-6 mRNA expression in a dose-dependent manner. These results suggest that LPS, RsDPLA, and paclitaxel are recognized by the same receptor complex on ST2 cells, and that the receptor functions without membrane CD14. © Pearson Professionals Ltd 1997.
  • T ODAGIRI, K TOMINAGA, K TOBITA, S OHTA
    JOURNAL OF GENERAL VIROLOGY 75 43 - 53 1994年01月 [査読有り][通常論文]
     
    The mutated non-structural NS2 protein of an influenza A virus mutant, Wa-182, has been shown to be responsible for the production of defective interfering (DI) particles lacking the PA gene after a single cycle high-multiplicity infection. Using a subclone of Wa-182, A3/e-3, that inherited the Wa-182 phenotype but contained only a marginal amount of DI RNAs derived from the PA gene, we showed that replication of the PA genome RNA was suppressed primarily at the step of complementary RNA (cRNA) synthesis. On the other hand, the small amounts of DI RNA species present in the stock of A3/e-3 were shown to be replicated efficiently. These findings suggested that the suppression of cRNA synthesis of the PA gene was caused by preferential amplification of the DI RNAs. The suppression of PA gene cRNA synthesis subsequently resulted in suppression of both virion RNA synthesis and secondary transcription of the PA gene. Such aberrant replication of the PA gene was found to be attributable to an amino acid change in the NS2 protein at position 32, from isoleucine to threonine. These results suggest that the NS2 protein plays a role in promoting normal replication of the genomic RNAs by preventing the replication of short-length RNA species.
  • K TOMINAGA, J HAYASHI, Y KAGAWA, S OHTA
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 194 1 544 - 551 1993年07月 [査読有り][通常論文]
  • K TOMINAGA, S AKIYAMA, Y KAGAWA, S OHTA
    BIOCHIMICA ET BIOPHYSICA ACTA 1131 2 217 - 219 1992年06月 [査読有り][通常論文]
     
    A genomic gene for human mitochondrial transcription factor 1 was cloned from a human genomic library and its 5' flanking region was sequenced. No typical TATA and three consensus sequences for potential Sp1 binding site were found in its 5' flanking region of 2 kilobase pairs. There were, at least, four common sequences among some nuclear genes for mitochondria-related proteins.
  • Y YAMAGUCHI, T SUDA, S OHTA, K TOMINAGA, Y MIURA, T KASAHARA
    BLOOD 78 10 2542 - 2547 1991年11月 [査読有り][通常論文]
  • Y KOBAYASHI, MY MOMOI, K TOMINAGA, H SHIMOIZUMI, K NIHEI, M YANAGISAWA, Y KAGAWA, S OHTA
    AMERICAN JOURNAL OF HUMAN GENETICS 49 3 590 - 599 1991年09月 [査読有り][通常論文]
     
    MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) is a major subgroup of heterogeneous mitochondrial diseases. For identifying a mutation in the mitochondrial gene, we isolated, from the same muscle tissue from a patient with MELAS, cell lines with distinctly different phenotypes: one was respiration-deficient, and the other was apparently normal. Compared with the normal cells only one A-to-G nucleotide transition at nucleotide 3243 in the tRNA-Leu (UUR) gene was found in whole mtDNA of the respiration-deficient cells. This mutation was also found in eight patients, from unrelated families, who had MELAS in a heteroplasmic manner but was not found in control individuals. Therefore, the single point mutation causes the functional abnormality in the respiratory chain of mitochondria.
  • S OHTA, K TOMINAGA, Y KAGAWA, H TOMURA, H ENDO
    MITOCHONDRIAL ENCEPHALOMYOPATHIES 7 103 - 112 1991年 [査読有り][通常論文]
  • Y KAGAWA, M YUZAKI, S OHTA, K TOMINAGA, M OTSUKA, KANAZAWA, I
    MITOCHONDRIAL ENCEPHALOMYOPATHIES 7 129 - 139 1991年 [査読有り][通常論文]
  • Y KOBAYASHI, MY MOMOI, K TOMINAGA, T MOMOI, K NIHEI, M YANAGISAWA, Y KAGAWA, S OHTA
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 173 3 816 - 822 1990年12月 [査読有り][通常論文]

MISC

共同研究・競争的資金等の研究課題

  • エピジェネティック制御因子による神経幹細胞の維持機構および神経分化に関する研究
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 冨永 薫
     
    幹細胞の存在が中枢神経系を含む多くの組織で報告されており、その自己複製能と分化能の厳密な調節を通じて組織の恒常性維持が保たれている。神経幹細胞は中枢神経系を構成するニューロンとグリアに分化できる多分化能を持つ細胞で、継続的に機能的な神経細胞を個体の一生を通じて供給している。神経系の構築にはエピジェネティックな調節機構が関与し、その破綻が神経幹細胞の維持や神経発生、高次脳機能に大きな影響を与える。しかしながら、エピジェネティック因子により、これらの事象がどのように制御されるのかについての詳細な分子機構は、未だ不明である。本研究では、神経幹細胞特異的Tip60欠損マウスを用いて、神経幹細胞の自己複製および神経分化におけるヒストンアセチル化の役割を明らかにすることを目的とする。当該年度は主にニューロスフェア法によるin vitroの解析を確立し、以下の結果を得た。 1、胎生期14.5日の神経幹細胞特異的Tip60欠損マウス脳の大脳皮質より神経幹前駆細胞を分離し、ニューロスフェア法による培養系を確立した。 2、神経幹細胞特異的Tip60欠損マウス胎児脳由来の神経幹前駆細胞は、増殖能が著しく低下しており、小頭症を引き起こす一因であると考えられた。 3、神経幹細胞特異的Tip60欠損マウス脳由来の神経幹前駆細胞にBrdUを取り込ませ、細胞増殖率を比較検討した結果、神経幹細胞特異的Tip60欠損マウス胎児由来の神経幹前駆細胞では、BrdUの取り込み率が低く、細胞増殖の低下が確認された。 4、神経幹細胞特異的Tip60欠損マウス胎児脳由来の神経幹前駆細胞を分化誘導培地で処理したところ、神経分化が著しく低下していた。対照的にアストロサイトへの分化は促進されており、Tip60ヒストンアセチル化酵素が、神経分化とグリア細胞分化の両方に関与することが明らかとなった。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2012年04月 -2016年03月 
    代表者 : 冨永 薫
     
    本研究は、クロモドメインを持つクロマチン結合タンパク質MRG15の神経幹/前駆細胞における役割を解明することを目的とした。神経幹/前駆細胞特異的MRG15欠損マウスは、正常に生まれてきたが、成長にともなう体重の増加がコントロール群に比べ、著しく抑制されていた。神経幹/前駆細胞特異的MRG15欠損マウスから分離された神経幹/前駆細胞は、その数が少ないだけでなく、増殖も抑制されていた。MRG15を介した遺伝子発現制御やスプライシング機構が、神経幹/前駆細胞の維持および機能に重要な役割を担っていることが明らかとなった。
  • スカベンジャー受容体の感染防御における役割の解明
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 1996年 -1996年 
    代表者 : 中野 昌康, 富永 薫, 切替 照雄, 紀伊 道里
     
    スカンベンジャー受容体欠損マウスは、129/SvJマウス由来のES細胞を相同組換えによってスカンベンジャー受容体遺伝子を不活化することによって共同研究者の児玉龍彦博士らによって作成された。ES細胞をC57BL/6Jマウスブラストサイトに注入してキメラマウスを作成し、このキメラマウスをさらにICRマウスと交配し、ヘテロ接合体を得た。このヘテロ接合体の雄雌を交配させてホモ接合体であるスカベンジャー受容体欠損マウスを作成した。コントロールの正常マウスは、同様にヘテロ接合体のFlの正常ホモ接合体を用いた。これらのマウスは、2世代同腹交配した後、2つのグループ(ストックAとストックB)にわけて、異なった2施設で4から5世代交配させている。スカベンジャー受容体欠損マウスの腹腔マクロファージをスカベンジャー受容体に対する抗体で染色すると、正常マウスではスカベンジャー受容体タンパク質が強く発現していたが、ノックアウトマウスでは全く発現していなかった。そこでこれらの培養マクロファージをLPSで刺激した時に誘導されるサイトカイン産生を観察した。種々の濃度のLPSで刺激した場合、TNF、IL-1β、IL-6の産生の用量依存性は正常マクロファージとスカベンジャー受容体欠損マクロファージとでほとんど変化がみられなかった。LPS刺激によるNO産生をみると、ストックAのノックアウトマウスではNO産生量の低下がみられたが、ストックBでは、逆にNO産生がそれぞれのコントロールより亢進していた。同様なストックによる表現系の違いは、リステリア菌感染でも観察された。この場合、NO産生の違いなどを左右する因子は、スカベンジャー遺伝子ではなく、他の遺伝子の関与が疑われた。 マクロファージを酸化LDLやデキストラン硫酸であらかじめ処理すると、LPSやINFγ刺激によって誘導されるNOやIL-lβ産生が低下した。そこでノックアウトマウスのマクロファージをデキストラン硫酸で処理しLPSで刺激をした。コントロールマクロファージでは、用いたデキストラン硫酸の濃度に依存して、NO産生量が低下した。一方、スカベンジャー受容体欠損マクロファージもデキストラン硫酸の濃度に従って、NO産生が低下した。しかし、スカベンジャー受容体欠損マクロファージでのデキストラン硫酸の効果は、コントロールと比べて10倍以上弱かった。これらの結果は、LPS刺激によるNO産生がスカベンジャー受容体を介して制御されている可能性を示唆しているが、必ずしもマクロファージのスカベンジャー経路が単純ではないことが示唆された。このようなスカベンジャー受容体とLPSの結び付きの詳細はさらに検討される必要がある。
  • 日本学術振興会:科学研究費助成事業 奨励研究(A)
    研究期間 : 1996年 -1996年 
    代表者 : 冨永 薫
     
    昨年度我々が作製したLPS応答性胎児線維芽細胞株(MEF.He)およびLPS低応答性胎児線維芽細胞株(MEF.HeJ)を、LPSを含む種々の刺激物質で刺激し、IL-6の発現を指標に、両細胞株の応答性を調べた。MEF.He細胞株では、LPSのみならずマウスマクロファージにおいてLPSと同様な作用を示すことが明らかとなっているタキソ-ルや細胞内セカンドメッセンジャーの一つであるセラミドに応答してIL-6mRNAを発現した。一方、MEF.HeJ細胞株では、LPSのみならずこれらの刺激に不応答で、IL-6mRNAを発現しなかった。陽性コントロールのIL-1α刺激では、両細胞株共に応答し、IL-6mRNAを発現した。MEFは、マクロファージに発現し、LPSレセプターの一つであると考えられているCD14分子を発現していないので、CD14を介さないLPSシグナル伝達系の存在が示唆された。また、チロシンキナーゼ阻害剤を用いた実験から、MEF.HeにおけるLPSシグナル伝達系にもチロシンキナーゼが関与することを明らかにし、MEFのLPSシグナル伝達系はマクロファージのそれと共通性が高いことが示唆された。 LPSで細胞を前処理すると、2回目のLPS刺激に対して著しく不応答になることが知られており、LPSトレランスとよばれている。マウス腹腔マクロファージを用い、LPSトレランス状態にある細胞のLPSシグナル伝達系を調べた。LPSレランスマクロファージでは、3種のMAPキナーゼ(MAPK,p38,JNK)全ての活性化が抑制されていた。また、MAPKの上流に位置するキナーゼであるRaf-1の活性化も抑制されており、これらMAPキナーゼファミリーの上流のキナーゼがLPSトレランスでは抑制されることを明らかにした。またLPS刺激により活性化される重要な転写因子NF-κBの活性化もLPSトレランスマクロファージでは抑制されていた。以上の結果は、LPSトレランスがLPSシグナル伝達系を構成するかなり上流に位置する分子の抑制により引き起こされることを強く示唆していた。
  • 日本学術振興会:科学研究費助成事業 奨励研究(A)
    研究期間 : 1995年 -1995年 
    代表者 : 富永 薫
     
    C3H/HeJマウスは、リポ多糖(LPS)に著しく低応答性であり、このマウスのLPS低応答性に関わる変異分子は第4染色体上にコードされると考えられている。C3H/HeJマウスのLPS低応答性の機構解明および変異分子の同定を目的とし、C3H/HeJマウスの胎児線維芽細胞のLPS応答性を解析すると共に、その細胞株樹立を試みた。コントロールとして同系のC3H/Heマウスを用いた。 C3H/Heマウス由来の胎児繊維芽細胞は、LPSに応答し、インターロイキン-6(IL-6)を産生した。その産生量はチオグリコレート培地で誘導した同マウスの腹腔マクロファージに比べ低かったが、1ng/ml程度の低濃度のLPSにも応答し、IL-6を産生した。一方、C3H/HeJマウス由来の胎児線維芽細胞は、低濃度のLPS刺激ではIL-6を産生せず、LPSに著しく低応答性であった。高濃度のLPOS刺激(1μg/ml以上)では、C3H/HeJマウス由来線維芽細胞でもIL-6を産生した。これは、再精製したLPSでも認められたので、IL-6の産生性に起因するものと考えられた。両線維芽細胞に複製開始点を欠くSV40ウイルスDNAをを導入し、細胞株を樹立した。これらの細胞株は、初代培養線維芽細胞に比べIL-6の産生性は幾分落ちるものの、ほど同様のLPS応答性を示した。樹立された細胞株は、マクロファージにおけるLPS/LPS結合タンパク質のレセプターであるCD14分子に対するmRNAを発現しておらず、膜結合型CD14に依存しないLPS認識機構の存在が推定された。また、^<125>Iで標識したLPSを用いてLPSの結合能を両細胞株で調べたが、差は認められなかった。従って、C3H/HeJマウスの変異分子はLPSの結合以降に位置すると考えられた。C3H/HeJマウスの変異分子の同定およびLPSのシグナル伝達系の解析に有用であると考えられる細胞株が樹立された。


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