Updated on 2021/12/28

写真a

 
OKAMOTO Kuniaki
 
Organization
Medicine, Dentistry and Pharmaceutical Sciences Professor
Position
Professor
External link

Degree

  • (BLANK) ( Kyushu University )

Research Interests

  • osteoclast

  • oral cancer

  • intercellular transport

Research Areas

  • Life Science / Oral biological science

Professional Memberships

▼display all

 

Papers

  • Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells. Reviewed International journal

    Namba Y, Sogawa C, Okusha Y, Kawai H, Itagaki M, Ono K, Murakami J, Aoyama E, Ohyama K, Asaumi JI, Takigawa M, Okamoto K, Calderwood SK, Kozaki KI, Eguchi T

    Front Oncol.   8   376 - 376   2018.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10-35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

    File: 72. Namba (Frontiers in Oncol).pdf

    DOI: 10.3389/fonc.2018.00376

    PubMed

    researchmap

  • Anti-EGFR antibody cetuximab is secreted by oral squamous cell carcinoma and alters EGF-driven mesenchymal transition. Reviewed International journal

    Toshifumi Fujiwara, Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Jun Murakami, Soichiro Ibaragi, Jun-Ichi Asaumi, Kuniaki Okamoto, Stuart K Calderwood, Ken-Ichi Kozaki

    Biochemical and biophysical research communications   503 ( 3 )   1267 - 1272   2018.9

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

    File: 69. Fujiwara (BBRC).pdf

    DOI: 10.1016/j.bbrc.2018.07.035

    PubMed

    researchmap

  • HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells. Reviewed International journal

    Kisho Ono, Takanori Eguchi, Chiharu Sogawa, Stuart K Calderwood, Junya Futagawa, Tomonari Kasai, Masaharu Seno, Kuniaki Okamoto, Akira Sasaki, Ken-Ichi Kozaki

    Journal of cellular biochemistry   119 ( 9 )   7350 - 7362   2018.9

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90β, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

    File: 68. Ono (J Cell Biochem).pdf

    DOI: 10.1002/jcb.27039

    PubMed

    researchmap

  • 急速転移性癌細胞株由来細胞外小胞による破骨細胞分化および癌細胞浸潤の制御

    板垣 まみ, 十川 千春, 奥舎 有加, 江口 傑徳, 小野 喜章, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2018   347 - 347   2018.9

     More details

    Language:Japanese   Publisher:(一社)歯科基礎医学会  

    researchmap

  • Rutaecarpine attenuates osteoclastogenesis by impairing macrophage colony stimulating factor and receptor activator of nuclear factor κ-B ligand-stimulated signalling pathways. Reviewed International journal

    Yutaka Fukuma, Eiko Sakai, Shunsuke Komaki, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Clinical and experimental pharmacology & physiology   45 ( 8 )   863 - 865   2018.8

     More details

    Language:English  

    Rutaecarpine is a major alkaloid isolated from Evodia rutaecarpa. Here, we investigated the effects of rutaecarpine on osteoclast differentiation induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κ-B ligand (RANKL) in bone marrow-derived macrophages (BMMs). Treatment with rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of BMM-derived osteoclasts. Mechanistically, rutaecarpine decreased the protein level of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) and the phosphorylation of other signalling pathways during the osteoclast differentiation. Thus, rutaecarpine may be useful as a therapeutic agent for the treatment of bone diseases.

    DOI: 10.1111/1440-1681.12941

    PubMed

    researchmap

  • Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment Reviewed

    Takanori Eguchi, Chiharu Sogawa, Yuka Okusha, Kenta Uchibe, Ryosuke Iinuma, Kisho Ono, Keisuke Nakano, Jun Murakami, Manabu Itoh, Kazuya Arai, Toshifumi Fujiwara, Yuri Namba, Yoshiki Murata, Kazumi Ohyama, Manami Shimomura, Hirohiko Okamura, Masaharu Takigawa, Tetsuya Nakatsura, Ken-ichi Kozaki, Kuniaki Okamoto, Stuart K. Calderwood

    PLoS ONE   13 ( 2 )   2018.2

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Public Library of Science  

    Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

    DOI: 10.1371/journal.pone.0191109

    Scopus

    PubMed

    researchmap

  • Actin binding LIM 1 (abLIM1) negatively controls osteoclastogenesis by regulating cell migration and fusion. Reviewed International journal

    Haruna Narahara, Eiko Sakai, Yu Yamaguchi, Shun Narahara, Mayumi Iwatake, Kuniaki Okamoto, Noriaki Yoshida, Takayuki Tsukuba

    Journal of cellular physiology   234 ( 1 )   486 - 499   2018.1

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Actin binding LIM 1 (abLIM1) is a cytoskeletal actin-binding protein that has been implicated in interactions between actin filaments and cytoplasmic targets. Previous biochemical and cytochemical studies have shown that abLIM1 interacts and co-localizes with F-actin in the retina and muscle. However, whether abLIM1 regulates osteoclast differentiation has not yet been elucidated. In this study, we examined the role of abLIM1 in osteoclast differentiation and function. We found that abLIM1 expression was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation, and that a novel transcript of abLIM1 was exclusively expressed in osteoclasts. Overexpression of abLIM1 in the murine monocytic cell line, RAW-D suppressed osteoclast differentiation and decreased expression of several osteoclast-marker genes. By contrast, small interfering RNA-induced knockdown of abLIM1 enhanced the formation of multinucleated osteoclasts and markedly increased the expression of the osteoclast-marker genes. Mechanistically, abLIM1 regulated the localization of tubulin, migration, and fusion in osteoclasts. Thus, these results indicate that abLIM1 negatively controls osteoclast differentiation by regulating cell migration and fusion mediated via actin formation.

    File: 65. Narahara(J Cell Physiol).pdf

    DOI: 10.1002/jcp.26605

    PubMed

    researchmap

  • Rutaecarpine attenuates osteoclastogenesis by impairing M-CSF and RANKL-stimulated signaling pathways. Reviewed

    Fukuma Y, Sakai E, Komaki S, Nishishita K, Okamoto K, Tsukuba T

    Clin Exp Pharmacol Physiol.   2018

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    File: 66. Fukuma (Clin Exp Pharmacol Physiol.pdf

    researchmap

  • Transcription factor EB (TFEB) influences invasion and migration in oral squamous cell carcinomas. Reviewed International journal

    Sakamoto H, Yamashita K, Okamoto K, Kadowaki T, Umeda M, Tsukuba T

    Oral Dis.   24 ( 5 )   741 - 748   2018

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    OBJECTIVE: Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and plays an important role in various cancers. However, the function of TFEB in oral squamous cell carcinomas has not been examined. The aim of this study was to elucidate the role of TFEB in oral squamous cell carcinomas. MATERIALS AND METHODS: Expression levels of TFEB were examined in six different human oral squamous carcinoma cells: HSC2, HSC3, HSC4, SAS, OSC20, and SCC25. Knockdown of TFEB using small interfering RNA in HSC2 and HSC4 cells was performed. Cell morphology was observed by immunofluorescence microscopy. Cell proliferation, invasion, and adhesion were analyzed. RESULTS: Expression levels of TFEB were high in HSC2, moderate in HSC4 and SCC25, and low in HSC3 and OSC20 cells. Knockdown of TFEB did not affect proliferation of HSC2 and HSC4 cells, but did induced enlargement of lysosomes and endosomes in HSC4 cells. TFEB silencing reduced invasion and migration of these HSC cell squamous carcinoma cells; however, increased cell adhesion was also observed. CONCLUSION: TFEB knockdown reduces invasion and migration of cancer cells, likely through lysosomal regulation. Taken together, TFEB influences cell invasion and migration of oral squamous cell carcinomas.

    File: 63. Sakamoto (Oral Dis).pdf

    DOI: 10.1111/odi.12826

    PubMed

    researchmap

  • The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells. Reviewed International journal

    Okusha Y, Eguchi T, Sogawa C, Okui T, Nakano K, Okamoto K, Kozaki KI

    J Cell Biochem.   119 ( 9 )   7363 - 7376   2018

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells.

    File: 67. Okusha (J Cell Biochem).pdf

    DOI: 10.1002/jcb.27040

    PubMed

    researchmap

  • Dihydroartemisinin represses osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα. Reviewed

    Shunsuke Komaki, Eiko Sakai, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Biomedical research (Tokyo, Japan)   39 ( 4 )   169 - 177   2018

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Osteoclasts are multinucleated bone resorbing cells whose differentiation is regulated by several important signaling pathways. Several lines of evidence indicate that dihydroartemisinin (DHA), an anti-malarial drug, inhibits osteoclast differentiation with little cytotoxicity. However, the detailed inhibitory mechanisms of DHA on osteoclastogenesis from native cells remain to be elucidated. In this study, we investigated the effects of DHA on the differentiation of bone marrow-derived macrophages into osteoclasts. DHA inhibited receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclast formation and its bone resorbing activity. Mechanistically, DHA treatment markedly abolished phosphorylation of IκBα, and slightly affected a p38 MAPK dependent pathway. Moreover, DHA treatment induced down-regulation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for osteoclast differentiation and its target proteins, such as Src and cathepsin K. These results indicate that DHA represses RANKL-induced osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

    File: 70. Komaki (Biomed Res).pdf

    DOI: 10.2220/biomedres.39.169

    PubMed

    researchmap

  • 扁平上皮癌細胞の増殖能・浸潤能・転移能における転写因子TFEBの役割

    坂元 裕, 山下 健太郎, 岡元 邦彰, 門脇 知子, 梅田 正博, 筑波 隆幸

    生命科学系学会合同年次大会   2017年度   [1P - 1012]   2017.12

     More details

    Language:Japanese   Publisher:生命科学系学会合同年次大会運営事務局  

    researchmap

  • 異なる転移能を有する口腔扁平上皮癌細胞由来エクソソームに含まれるプロテオームの特性

    小野 喜章, 江口 傑徳, 十川 千春, 村上 純, 藤原 敏史, 笠井 智成, 妹尾 昌治, 佐々木 朗, 小崎 健一, 岡元 邦彰

    生命科学系学会合同年次大会   2017年度   [1LBA - 052]   2017.12

     More details

    Language:Japanese   Publisher:生命科学系学会合同年次大会運営事務局  

    researchmap

  • The Rho-specific guanine nucleotide exchange factor Plekhg5 modulates cell polarity, adhesion, migration, and podosome organization in macrophages and osteoclasts. Reviewed International journal

    Mayumi Iwatake, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Experimental cell research   359 ( 2 )   415 - 430   2017.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Osteoclasts are multinucleated bone-resorbing cells that are formed by fusion of monocyte/macrophage lineage. Osteoclasts and macrophages generate podosomes that are actin-based dynamic organelles implicated in cell adhesion, spreading, migration, and degradation. However, the detailed mechanisms of podosome organization remain unknown. Here, we identified the Rho-specific guanine-nucleotide exchange factor (Rho-GEF) Plekhg5 as an up-regulated gene during differentiation of osteoclasts from macrophages. Knockdown of Plekhg5 with small interfering RNA in both macrophages and osteoclasts induced larger cell formation with impaired cell polarity and resulted in an elongated and flattened shape. In macrophages, Plekhg5 depletion enhanced random migration, but impaired directional migration, adhesion, and matrix degradation. Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption. Plekhg5 depletion affected signaling and localization of several Rho downstream effectors. In fact, end-binding protein 1 (EB1), cofilin and vinculin were abnormally localized in Plekhg5-depleted cells, and mDia1 and LIM kinase (LIMK)1 were upregulated in Plekhg5-depleted cells compared with control cells. However, overexpression of Plekhg5 in macrophages induced an increase in its mRNA level, but failed to increase the protein level, indicating that overexpressed Plekhg5 was degraded in macrophages but not HEK293T cells. Thus, Plekhg5 affects cell polarity, migration, adhesion, degradation, and podosome organization in macrophages and osteoclasts.

    File: 62. Iwatake (Exp Cell Res).pdf

    DOI: 10.1016/j.yexcr.2017.08.025

    PubMed

    researchmap

  • Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis Reviewed

    Eiko Sakai, Masanobu Morita, Masahiro Ohuchi, Mizuho A. Kido, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Ken Itoh, Masayuki Yamamoto, Takayuki Tsukuba

    FASEB JOURNAL   31 ( 9 )   4011 - 4022   2017.9

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:FEDERATION AMER SOC EXP BIOL  

    Kelch-like ECH-associated protein 1 (Keap1) binds to nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for antioxidant enzymes, to suppress Nrf2 activation. The role of oxidative stress in many diseases supports the possibility that processes that are associated with Nrf2 activation might offer therapeutic potential. Nrf2 deficiency induces osteoclastogenesis, which is responsible for bone loss, by activating receptor activator of NF-kappa B ligand (RANKL)-mediated signaling; however, the effects of Keap1 deficiency remain unclear. By using Keap1-deficient newborn mice, we observed that talus and calcaneus bone formation was partially retarded and that osteoclast number was reduced in vivo without severe gross abnormalities. In addition, Keap1-deficient macrophages were unable to differentiate into osteoclasts in vitro via attenuation of RANKL-mediated signaling and expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor that is involved in osteoclastogenesis. Furthermore, Keap1 deficiency up-regulated the expression of Mafb, a negative regulator of NFATc1. RANKL-induced mitochondrial gene expression is required for down-regulation of IFN regulatory factor 8 (IRF-8), a negative transcriptional regulator of NFATc1. Our results indicate that Keap1 deficiency down-regulated peroxisome proliferator-activated receptor-gamma coactivator 1 beta and mitochondrial gene expression and up-regulated Irf8 expression. These results suggest that the Keap1/Nrf2 axis plays a critical role in NFATc1 expression and osteoclastogenic progression.

    File: 60. Sakai (FASEB ).pdf

    DOI: 10.1096/fj.201700177R

    Web of Science

    researchmap

  • Rab44, a novel large Rab GTPase, negatively regulates osteoclast differentiation by modulating intracellular carcium levels followd by NFATc1 activation. Reviewed

    Yamaguchi Y, Sakai E, Okamoto K, Kajiya H, Okabe K, Naito M, Kadowaki T, Tsukuba T

    Cell Mol Life Sci.   75   33 - 48   2017.8

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    File: 61. Yamaguchi (Cell Mol Life Sci).pdf

    researchmap

  • The dental resin monomers HEMA and TEGDMA have inhibitory effects on osteoclast differentiation with low cytotoxicity Reviewed

    Hiroyuki Inamitsu, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Hiroshi Murata, Takayuki Tsukuba

    JOURNAL OF APPLIED TOXICOLOGY   37 ( 7 )   817 - 824   2017.7

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY  

    The dental resin monomers 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) are released from the resin matrix due to unpolymerized monomers; once released, they influence various biological functions and the viability of cells in the oral environment. Although HEMA and TEGDMA have various effects on cells, including inflammation, inhibition of cell proliferation or differentiation, and apoptosis, the effects of these monomers on osteoclasts remain unknown. In this study, we investigated the effects of HEMA and TEGDMA on osteoclast differentiation of bone marrow-derived macrophages or murine monocytic cell line RAW-D. Both HEMA and TEGDMA inhibited osteoclast formation and their bone-resorbing activity at non-cytotoxic concentrations. Moreover, HEMA and TEGDMA decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of osteoclast differentiation, and of osteoclast markers that are transcriptionally regulated by NFATc1, including Src and cathepsin K. Regarding their effects on signaling pathways involved in osteoclast differentiation, HEMA impaired the phosphorylation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas TEGDMA attenuated the phosphorylation of Akt and Jun N-terminal kinase. Thus, HEMA and TEGDMA inhibit osteoclast differentiation through different signaling pathways. This is the first report on the effects of the monomers HEMA and TEGDMA on osteoclasts. Copyright (C) 2017 John Wiley & Sons, Ltd.

    File: 58. Inamitsu(J Appl Toxicol.pdf

    DOI: 10.1002/jat.3429

    Web of Science

    researchmap

  • New functions of lysosomes in bone cells

    Takayuki Tsukuba, Eiko Sakai, Kazuhisa Nishishita, Tomoko Kadowaki, Kuniaki Okamoto

    Journal of Oral Biosciences   59   92 - 95   2017.5

     More details

    © 2017 Japanese Association for Oral Biology Background Lysosomes are intracellular acidic organelles that contain approximately 50 hydrolases and 25 species of integral membrane proteins. Although lysosome-like specific compartments, termed lysosome-related organelles (LROs), are found in osteoclasts, their functions in these cells and lysosomal functions in osteoblasts remain to be elucidated. Highlight Recently, we found that expression of RAB27A is markedly increased during osteoclastic differentiation. RAB27A deficiency causes multinucleation and giant cell formation, characterized by abnormal transport of cell surface receptors and LROs into osteoclasts. Furthermore, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. Overexpression of TFEB in preosteoblastic MC3T3-E1 cells enhances osteoblastic differentiation via decreased expression of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). These results indicate that the expression of ATF4 and CHOP is essential for differentiation into osteoblasts. Conclusion RAB27A participates in bone resorption by LROs in osteoclasts. In addition, lysosomal biogenesis modulated by TFEB is necessary for osteoblastic differentiation.

    DOI: 10.1016/j.job.2017.01.004

    Scopus

    researchmap

  • Cathepsin K modulates invasion, migration and adhesion of oral squamous cell carcinomas invitro Reviewed

    K. Yamashita, M. Iwatake, K. Okamoto, S-i Yamada, M. Umeda, T. Tsukuba

    ORAL DISEASES   23 ( 4 )   518 - 525   2017.5

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY  

    ObjectiveCathepsin K was initially discovered as an osteoclast-specific cysteine proteinase, but the enzyme is also expressed in various cancers including oral squamous cell carcinomas. This study aimed to clarify the function of cathepsin K in oral squamous cell carcinomas.
    Materials and MethodsExpression levels of cathepsin K were examined in six types of cell carcinomas. Carcinomas overexpressing cathepsin K were constructed. Effects of cathepsin K overexpression and treatment with odanacatib, a specific cathepsin K inhibitor, on cell invasion, migration and adhesion were analysed.
    ResultsDifferent levels of cathepsin K were expressed in carcinomas. Cathepsin K was predominantly localised in lysosomes. Cathepsin K overexpression impaired the proliferation of carcinomas. Invasion analysis showed that cathepsin K overexpression enhanced invasion and migration of carcinomas, whereas inhibition of cathepsin K by odanacatib caused the opposite effects in carcinomas. Cathepsin K overexpression also increased cell adhesion and slightly increased surface expression of the adhesion receptor CD29/integrin (1).
    ConclusionsThe enhanced invasion of carcinomas resulting from cathepsin K overexpression is probably due to the increased cell migration and adhesion. Thus, cathepsin K is implicated not only in protein degradation but also in invasion, migration and adhesion of oral squamous cell carcinomas.

    File: 59. Yamashita (Oral Dis).pdf

    DOI: 10.1111/odi.12643

    Web of Science

    researchmap

  • Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-kappa B ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-alpha-induced osteoclast formation in vivo Reviewed

    Eiko Sakai, Yuri Aoki, Masako Yoshimatsu, Kazuhisa Nishishita, Mayumi Iwatake, Yutaka Fukuma, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    PHYTOMEDICINE   23 ( 8 )   828 - 837   2016.7

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER GMBH, URBAN & FISCHER VERLAG  

    Background: Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated.
    Purpose: To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption.
    Methods: Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-alpha (TNF-alpha)-mediated osteoclastogenesis was examined in vivo.
    Results: R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-kappa B alpha (I kappa B alpha) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-kappa B in vitro, as well as TNF-alpha-mediated osteoclastogenesis in vivo.
    Conclusions: Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction. (C) 2016 Elsevier GmbH. All rights reserved.

    File: 57. Sakai (Phytomedicine) .pdf

    DOI: 10.1016/j.phymed.2016.04.002

    Web of Science

    researchmap

  • The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway Reviewed

    Erika Yoneshima, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Noriaki Yoshida, Takayuki Tsukuba

    JOURNAL OF CELLULAR PHYSIOLOGY   231 ( 6 )   1321 - 1333   2016.6

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway. (c) 2015 Wiley Periodicals, Inc.

    File: 56. Yoneshima (J Cellular Physiol).pdf

    DOI: 10.1002/jcp.25235

    Web of Science

    researchmap

  • Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation Reviewed

    Kaho Uchino, Kuniaki Okamoto, Eiko Sakai, Erika Yoneshima, Mayumi Iwatake, Yutaka Fukuma, Kazuhisa Nishishita, Takayuki Tsukuba

    PHYTOTHERAPY RESEARCH   29 ( 11 )   1714 - 1721   2015.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant-derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin-treated murine osteoblastic MC3T3-E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone-resorption activity through slightly decreased the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and inhibitor of nuclear factor kappa B; however, the phosphorylation of Akt and p38 slightly increased in bone marrow-derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T-cell cytoplasmic-1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases. Copyright (C) 2015 John Wiley & Sons, Ltd.

    File: 55. Uchino (Phytother res).pdf

    DOI: 10.1002/ptr.5416

    Web of Science

    researchmap

  • Punicalagin attenuates osteoclast differentiation by impairing NFATc1 expression and blocking Akt- and JNK-dependent pathways Reviewed

    Mayumi Iwatake, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    MOLECULAR AND CELLULAR BIOCHEMISTRY   407 ( 1-2 )   161 - 172   2015.9

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER  

    Punicalagin is a bioactive polyphenol that is classified as an ellagitannin. Although punicalagin has been shown to have various pharmacological effects, such as anti-oxidative, anti-inflammatory, and anti-tumor effects, no studies have reported the effects of punicalagin on osteoclasts (OCLs). In this study, we investigated the effects of punicalagin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand in the murine monocytic RAW-D cell line and bone marrow-derived macrophages (BMMs). Treatment with punicalagin significantly inhibited OCL formation from RAW-D cells and BMMs and prevented bone resorption of BMM-derived OCLs. Moreover, punicalagin impaired multinucleation and actin-ring formation in OCLs, and decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator of OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of punicalagin on intracellular signaling during the OCL differentiation of BMMs indicated that punicalagin-treated OCLs displayed markedly reduced phosphorylation of Jun N-terminal kinase and Akt, and partially impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and inhibitor of nuclear factor kappa-B alpha compared with untreated OCLs. Thus, punicalagin may affect bone metabolism by inhibiting OCL differentiation.

    File: 53. Iwatake (Mol Cell Biochem).pdf

    DOI: 10.1007/s11010-015-2466-3

    Web of Science

    researchmap

  • Cobalt protoporphyrin represses osteoclastogenesis through blocking multiple signaling pathways Reviewed

    Yuka Yashima, Kuniaki Okamoto, Eiko Sakai, Mayumi Iwatake, Yutaka Fukuma, Kazuhisa Nishishita, Takayuki Tsukuba

    BIOMETALS   28 ( 4 )   725 - 732   2015.8

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER  

    Cobalt protoporphyrin (CoPP) is a metallo-protoporphyrin that works as a powerful inducer of heme oxigenase-1 (HO-1) in various tissues and cells. Our recent studies have demonstrated that induction of HO-1 by several reagents inhibited differentiation and activation of osteoclasts (OCLs), which are multinucleated bone resorbing cells. However, the effects of CoPP on osteoclastogenesis remain to be elucidated. In this study, we report that CoPP inhibits receptor activator of nuclear factor kappa B ligand (RANKL)-induced OCL formation in a dose dependent manner. Importantly, CoPP had little cytotoxicity, but rather enhanced cell proliferation of OCLs. CoPP suppressed the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) as well as those of OCLs markers such as Src and cathepsin K, which are transcriptionally regulated by NFATc1 in mature OCLs. Western blot analyses also showed that CoPP abolished RANKL-stimulated phosphorylation of several major signaling pathways such as I kappa B, Akt, ERK, JNK and p38 MAPKs in OCL precursor cells. Thus, our results show that CoPP represses osteoclastogenesis through blocking multiple signaling pathways.

    File: 51. Yashima (Biometals).pdf

    DOI: 10.1007/s10534-015-9861-9

    Web of Science

    researchmap

  • Cafestol has a weaker inhibitory effect on osteoclastogenesis than kahweol and promotes osteoblast differentiation Reviewed

    Yutaka Fukuma, Eiko Sakai, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    BIOFACTORS   41 ( 4 )   222 - 231   2015.7

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Bone homeostasis is regulated by a balance between osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-mediated bone formation. Thus, developing a compound that simultaneously inhibits OCL function and promotes OBL function would be useful as a new medical therapy for bone diseases. Here, we examined the effects of cafestol, a coffee diterpene, on the differentiation of OCLs and OBLs. Cafestol prevented OCL formation in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Interestingly, the viability of OCLs treated with 10-50 mu M cafestol was significantly higher than that of untreated cells. At the molecular level, cafestol markedly decreased RANKL-induced phosphorylation of extracellular signal-regulated kinase (Erk) and inhibitor of nuclear factor kappa B alpha (IB). Compared to kahweol, another coffee-specific diterpene, the inhibitory effects of cafestol were milder on OCL differentiation, and cafestol and kahweol showed different characteristics in induction of the phase antioxidant enzymes and sensitivities in nuclear factor-erythroid 2-related factor 2 (Nrf2)-deficient BMMs. In addition to inhibiting OCLs, cafestol enhanced the differentiation of osteoblastic cells by increasing the mRNA levels of differentiation markers. Thus, cafestol inhibits OCL differentiation and promotes OBL differentiation, suggesting that cafestol may be a novel agent for bone diseases. (c) 2015 BioFactors, 41(4):222-231, 2015

    File: 54. Fukuma (BioFactors).pdf

    DOI: 10.1002/biof.1218

    Web of Science

    researchmap

  • Castalagin Exerts Inhibitory Effects on Osteoclastogenesis Through Blocking a Broad Range of Signaling Pathways with Low Cytotoxicity Reviewed

    Mayumi Iwatake, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    PHYTOTHERAPY RESEARCH   29 ( 6 )   917 - 924   2015.6

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Castalagin is a rare plant polyphenol that is classified as a hydrolyzable tannin. Although it has antioxidant, antitumorigenic, and leishmanicidal effects, the utility of castalagin against bone diseases remain to be elucidated. Here, we investigated the effects of castalagin on the differentiation of osteoclasts (OCLs), multinucleated bone-resorbing cells. After stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL), the formation of OCLs from bone marrow-derived macrophages was significantly inhibited by castalagin even at 1M. However, castalagin displayed little cytotoxicity at a higher concentration of 50M. The effects of castalagin on intracellular signaling during OCL differentiation showed that castalagin suppresses RANKL-stimulated phosphorylation of major signaling pathways including protein kinase B (Akt), extracellular signal-regulated kinase, Jun N-terminal kinase, p38 mitogen-activated protein kinases, and inhibitor of nuclear factor kappa B alpha. Moreover, following castalagin treatment, the protein levels of nuclear factor of activated T-cells, cytoplasmic 1, a master regulator for OCL differentiation, and NF-B were decreased. Thus, castalagin exerts inhibitory effects on osteoclastogenesis through blockage of a broad range of signaling pathways, but has low cytotoxicity. Copyright (c) 2015 John Wiley & Sons, Ltd.

    File: 50. Iwatake (Phytother res).pdf

    DOI: 10.1002/ptr.5333

    Web of Science

    researchmap

  • Rab27A Regulates Transport of Cell Surface Receptors Modulating Multinucleation and Lysosome-Related Organelles in Osteoclasts Reviewed

    Megumi Shimada-Sugawara, Eiko Sakai, Kuniaki Okamoto, Mitsunori Fukuda, Tetsuro Izumi, Noriaki Yoshida, Takayuki Tsukuba

    SCIENTIFIC REPORTS   5   2015.4

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Rab27A regulates transport of lysosome-related organelles (LROs) and release of secretory granules in various types of cells. Here, we identified up-regulation of Rab27A during differentiation of osteoclasts (OCLs) from bone-marrow macrophages (BMMs), by DNA microarray analysis. Rab27A deficiency in OCLs, using small interfering RNA (siRNA) knockdown in RAW-D cell line or BMMs derived from ashen mice, which display genetic defects in Rab27A expression, induced multinucleated and giant cells. Upon stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), essential cytokines for OCL differentiation, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosine-protein kinase (Src), and p-38 were slightly enhanced in ashen BMMs than in wild-type BMMs. The cell surface level of c-fms, an M-CSF receptor, was slightly higher in ashen BMMs than in wild-type BMMs, and down-regulation of RANK, a RANKL receptor, was delayed. In addition to receptors, OCLs derived from ashen mice exhibited aberrant actin ring formation, abnormal subcellular localization of lysosome-associated membrane protein (LAMP2) and cathepsin K (CTSK), and marked reduction in resorbing activity. Thus, these findings suggest that Rab27A regulates normal transport of cell surface receptors modulating multinucleation and LROs in OCLs.

    File: 51. Shimada-Sugawara (Sci Rep).pdf

    DOI: 10.1038/srep09620

    Web of Science

    researchmap

  • Cathepsin E: An Aspartic Protease with Diverse Functions and Biomedical Implications Reviewed

    K. Yamamoto, K. Okamoto, T. Tsukuba

    Encyclopedia of Cell Biology   1   681 - 690   2015.1

     More details

    Language:English   Publishing type:Part of collection (book)   Publisher:Elsevier Inc.  

    Aspartic peptidases are widely distributed among vertebrates, yeast, plants, fungi, bacteria, and viruses. Of these, the A1 family members, including cathepsin E and ß-secretase, are involved in specific and/or nonspecific degradation of proteins and peptides in intra- and/or extracellular spaces. In the last decade, cathepsin E and ß-secretase have received enormous interest because of their involvement in important biological processes and the close association of their abnormal expression and uncontrolled activity with human diseases. This review summarizes the current knowledge on biochemical properties and functions of cathepsin E and highlights the pathophysiological conditions caused by its deficiency.

    DOI: 10.1016/B978-0-12-394447-4.10078-1

    Scopus

    researchmap

  • Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet Reviewed

    Tomoko Kadowaki, Mizuho A. Kido, Junko Hatakeyama, Kuniaki Okamoto, Takayuki Tsukuba, Kenji Yamamoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   446 ( 1 )   212 - 217   2014.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE(-/-)) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE(-/-) mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE(-/-) mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE(-/-) mice. In fat-induced CatE(-/-) mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE(-/-) mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia. (C) 2014 Elsevier Inc. All rights reserved.

    File: 47. Kadowaki (BBRC).pdf

    DOI: 10.1016/j.bbrc.2014.02.089

    Web of Science

    researchmap

  • Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer Reviewed

    Tomoyo Kawakubo, Atsushi Yasukochi, Tatsuya Toyama, Satoru Takahashi, Kuniaki Okamoto, Takayuki Tsukuba, Seiji Nakamura, Yasuhiko Ozaki, Koichi Nishigaki, Hiroko Yamashita, Kenji Yamamoto

    CARCINOGENESIS   35 ( 3 )   714 - 726   2014.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remain unacceptably high. Therefore, more convenient diagnostic and prognostic methods still required to optimize treatments among the patients. Here, we report the clinical significance of the serum cathepsin E (CatE) activity as a novel prognostic marker for BC. Correlation analysis between the serum levels of CatE expression and clinicopathological parameters revealed that the activity levels, but not the protein levels, were negatively associated with the stages and progression of BC. Univariate and multivariate analyses demonstrated that the serum CatE activity was significantly correlated with favorable prognostic outcomes of the patients. The functional link of CatE expression to BC progression was further corroborated by in vivo and in vitro studies with mice exhibiting different levels of CatE expression. Multiparous CatE(/) mice spontaneously developed mammary tumors concomitant with morphological transformation and altered growth characteristics of the mammary glands. These alterations were associated in part with the induction of epithelialmesenchymal transition and the activation of -catenin-dependent pathway in mammary cells. Loss of CatE strongly induced the translocation and accumulation of Wnt5a in the nuclei, thereby leading to the aberrant trafficking, maturation and secretion of Wnt5a and the impaired signaling. The interaction of CatE and Wnt5a was verified by proximity ligation assay and by knockdown or restoration of CatE expression in the mammary cells. Consequently, our data demonstrate that CatE contributes to normal growth and development of mammary glands through proper trafficking and secretion of Wnt5a.

    File: 47. Kawakubo (Carcinogenesis).pdf

    DOI: 10.1093/carcin/bgt373

    Web of Science

    researchmap

  • Inhibitory effects of tert-butylhydroquinone on osteoclast differentiation via up-regulation of heme oxygenase-1 and down-regulation of HMGB1 release and NFATc1 expression Reviewed

    Yu Yamaguchi, Eiko Sakai, Hiroshi Sakamoto, Reiko Fumimoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF APPLIED TOXICOLOGY   34 ( 1 )   49 - 56   2014.1

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Our recent studies have shown that heme-oxygenase-1 (HO-1), a stress-induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert-butylhydroquinone (tBHQ), a pharmacological HO-1 inducer, on in vitro differentiation of bone marrow-derived macrophages (BMMs) or murine monocytic cell line RAW-D into OCLs. tBHQ inhibited the formation and the bone-resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (I kappa B alpha). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO-1/HMGB1 pathways. Copyright (c) 2012 John Wiley & Sons, Ltd.

    File: 48. Yamaguchi (J Appl Toxicol).pdf

    DOI: 10.1002/jat.2827

    Web of Science

    researchmap

  • Cobalt protoporphyrin prevents osteoclastogenesis via blocking of I kappa B phosphorylation Reviewed

    Yuka Yashima, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   124   232P - 232P   2014

     More details

    Language:English   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages Reviewed

    Takayuki Tsukuba, Michiyo Yanagawa, Tomoko Kadowaki, Ryosuke Takii, Yoshiko Okamoto, Eiko Sakai, Kuniaki Okamoto, Kenji Yamamoto

    PLOS ONE   8 ( 12 )   2013.12

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

    Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/-)) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/-) macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/-) macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/-) macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/-) cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/-) macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/-) macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

    File: 46. Tsukuba (Plosone).pdf

    DOI: 10.1371/journal.pone.0082415

    Web of Science

    researchmap

  • Structural and phylogenetic comparison of napsin genes: The duplication, loss of function and human-specific pseudogenization of napsin B Reviewed

    Kazuhisa Nishishita, Eiko Sakai, Kuniaki Okamoto, Takayuki Tsukuba

    GENE   517 ( 2 )   147 - 157   2013.4

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

    Aspartic proteinases form a widely distributed protein superfamily, including cathepsin D, cathepsin E, pepsins, renin, BACE and napsin. Human napsin genes are located on human chromosome 19q13, which comprises napsin A and napsin B. Napsin B has been annotated as a pseudogene because it lacks an in-frame stop codon; its nascent chains are cotranslationally degraded. Until recently, there have been no studies concerning the molecular evolution of the napsin protein family in the human genome. In the present study, we investigated the evolution and gene organization of the napsin protein family. Napsin B orthologs are primarily distributed in primates, while napsin A orthologs are the only napsin genes in other species. The corresponding regions of napsin B in the available sequences from primate species contain an in-frame stop codon at a position equivalent to that of human napsin A. In addition, a rare single-nucleotide polymorphism (SNP) that creates a proper stop codon in human napsin B was identified using HapMap populations. Recombinant protein expression and three-dimensional comparative modeling revealed that napsin B exhibits residual activity toward synthetic aspartic protease substrates compared with napsin A, presumably through a napsin B-specific Arg287 residue. Thus, napsin B was duplicated from napsin A during the early stages of primate evolution, and the subsequent loss of napsin B function during primate evolution reflected ongoing human-specific napsin B pseudogenization. (C) 2013 Elsevier B.V. All rights reserved.

    File: 44. NIshishita (Gene).pdf

    DOI: 10.1016/j.gene.2013.01.013

    Web of Science

    researchmap

  • Fisetin inhibits osteoclastogenesis through prevention of RANKLInduced ROS production by Nrf2-mediated Up-regulation of Phase II antioxidant enzymes Reviewed

    Eiko Sakai, Megumi Shimada-Sugawara, Yu Yamaguchi, Hiroshi Sakamoto, Reiko Fumimoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Journal of Pharmacological Sciences   121 ( 4 )   288 - 298   2013

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2-related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKLmediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes. © The Japanese Pharmacological Society.

    File: 45. Sakai (J Pharmacol Sci).pdf

    DOI: 10.1254/jphs.12243FP

    Scopus

    PubMed

    researchmap

  • Deltamethrin inhibits osteoclast differentiation via regulation of heme oxygenase-1 and NFATc1 Reviewed

    Hiroshi Sakamoto, Eiko Sakai, Reiko Fumimoto, Yu Yamaguchi, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    TOXICOLOGY IN VITRO   26 ( 6 )   817 - 822   2012.9

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PERGAMON-ELSEVIER SCIENCE LTD  

    Deltamethrin is a widely used pyrethroid pesticide. Although the cytotoxicity of deltamethrin has been reported, especially in neuronal cells, there is no information concerning the effects of deltamethrin on osteoclasts (OCLs). In this study, we showed that deltamethrin inhibited OCL differentiation in vitro. The effects of deltamethrin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated in bone marrow-derived macrophages (BMMs) or the murine monocytic cell line RAW-D. Treatment with deltamethrin inhibited OCL formation and bone resorption and up-regulated expression of heme oxygenase-1 (HO-1), an anti-oxidative stress enzyme. Deltamethrin also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator for OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of deltamethrin on intracellular signaling during the OCL differentiation of BMMs indicated that deltamethrin-treated OCLs displayed impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Jun N-terminal kinase, and Akt, and slightly delayed phosphorylation of inhibitor of nuclear factor kappa B alpha (I kappa beta alpha) compared with untreated OCLs. Thus, deltamethrin possibly affects bone metabolism by inhibiting OCL differentiation. (c) 2012 Elsevier Ltd. All rights reserved.

    File: 43. Sakamoto (Toxicol In Vitro).pdf

    DOI: 10.1016/j.tiv.2012.05.005

    Web of Science

    researchmap

  • The Coffee Diterpene Kahweol Prevents Osteoclastogenesis via Impairment of NFATc1 Expression and Blocking of Erk Phosphorylation Reviewed

    Reiko Fumimoto, Eiko Sakai, Yu Yamaguchi, Hiroshi Sakamoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118 ( 4 )   479 - 486   2012.4

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Osteoclasts (OCLs) are multinucleated bone resorbing cells whose differentiation is regulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). It is known that inflammatory cytokines and oxidative stress stimulate differentiation of OCLs. Here we evaluated the effects of kahweol, a coffee-specific diterpene, which has been reported to possess anti-oxidant and anti-inflammatory properties, on the differentiation of bone marrow derived macrophages (BMMs) or murine monocytic cell line RAW-D cells into OCLs. Kahweol dose-dependently inhibited the formation of tartrate-resistant acid phosphatase staining positive OCLs from both BMMs and RAW-D cells. In addition, kahweol prevented the bone resorbing activity of OCLs. Kahweol completely abolished RANKL-stimulated phosphorylation of extracellular signal-regulated kinase and impaired phosphorylation of Akt. Moreover, the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for OCL differentiation; and OCL markers transcriptionally regulated by NFATc1 such as Src and cathepsin K were down-regulated by kahweol treatment. As one of the molecular mechanisms for the inhibitory effects of kahweol, we also showed that kahweol up-regulated heme oxygenase-1 and inhibited high mobility group box I release. Thus, kahweol in coffee is a useful constituent for inhibition of OCL differentiation.

    File: 42. Fumimoto (J Pharmacol Sci).pdf

    DOI: 10.1254/jphs.11212FP

    Web of Science

    researchmap

  • Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice Reviewed

    Shun Narahara, Haruna Matsushima, Eiko Sakai, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    CELL AND TISSUE RESEARCH   348 ( 1 )   81 - 94   2012.4

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER  

    Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H2O2) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

    File: 41. Narahara (Cell Tissue Res).pdf

    DOI: 10.1007/s00441-012-1325-8

    Web of Science

    researchmap

  • Role of the transcription factor Sp1 in regulating the expression of the murine cathepsin E gene Reviewed

    Kuniaki Okamoto, Yoshiko Okamoto, Tomoyo Kawakubo, Jun-ichi Iwata, Yoshiyuki Yasuda, Takayuki Tsukuba, Kenji Yamamoto

    JOURNAL OF BIOCHEMISTRY   151 ( 3 )   263 - 272   2012.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Cathepsin E (CE) is an intracellular aspartic proteinase that is exclusively expressed in cells of the gastrointestinal tracts, lymphoid tissues, urinary organs and red blood cells. However, the molecular mechanism by which CE is predominantly expressed in these cells remains unknown. Here, we report the identification of several transcription start sites of the CE gene and their regulatory factors in gastric adenosarcoma cells. We first identified several unique transcription start sites in mouse CE genes by an oligo cap method. Their analysis also revealed the existence of a non-coding region similar to 24-kb upstream of exon 1 in the CE gene and also the existence of two transcripts for CE. Luciferase analyses in upstream of exon 1 revealed that this site contained putative binding regions for the transcription factors Sp1, AP-1 and cEts-1 essential for the expression of CE gene. Moreover, electrophoretic mobility shift assays revealed that the protein-oligonucleotides complex of the Sp1 site were supershifted by an anti-Sp1 antibody. The chromatin immunoprecipitation assay showed that Sp1 bound to the CE promoter region. In addition, overexpression of the Sp1 protein increased the expression of the CE protein. Altogether, these results suggest that Sp1 binding plays a particularly important role in the regulation of CE gene expression.

    File: 40. Okamoto (j Biochem).pdf

    DOI: 10.1093/jb/mvr135

    Web of Science

    researchmap

  • Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis Reviewed

    Eiko Sakai, Megumi Shimada-Sugawara, Kazuhisa Nishishita, Yutaka Fukuma, Mariko Naito, Kuniaki Okamoto, Koji Nakayama, Takayuki Tsukuba

    JOURNAL OF CELLULAR BIOCHEMISTRY   113 ( 2 )   486 - 498   2012.2

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor ?B ligand (RANKL) and macrophage colony-stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL-induced suppression of heme oxygenase-1 (HO-1), a heme-degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO-1 with hemin or curcumin in bone marrow-derived macrophages or RAW-D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen-activated protein kinase (MAPK) prevented the RANKL-mediated HO-1 suppression and extracellular release of HMGB1, these effects were p38 MAPK-dependent. Moreover, suppression of HO-1 in RAW-D cells by RNA interference promoted the activation of caspase-3 and HMGB1 release, whereas overexpression of HO-1 inhibited caspase-3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N-acetylcysteine abolished the HO-1/HMGB1/caspase-3 axis. These results suggest that RANKL-dependent HO-1 suppression leads to caspase-3 activation and HMGB1 release during osteoclastogenesis. J. Cell. Biochem. 113: 486498, 2012. (C) 2011 Wiley Periodicals, Inc.

    DOI: 10.1002/jcb.23372

    Web of Science

    researchmap

  • Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis Reviewed

    Eiko Sakai, Megumi Sugawara, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118 ( 2 )   208P - 208P   2012

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    File: 39. Sakai (J Cell Biochem).pdf

    DOI: 10.1002/jcb.23372

    Web of Science

    researchmap

  • Cathepsin E is critical for proper trafficking of cell surface proteins Reviewed

    Takayuki Tsukuba, Kuniaki Okamoto, Kenji Yamamoto

    Journal of Oral Biosciences   54 ( 1 )   48 - 53   2012

     More details

    Language:English   Publishing type:Research paper (international conference proceedings)   Publisher:Japanese Association for Oral Biology  

    Cathepsin E is an intracellular aspartic proteinase of the pepsin superfamily, which is predominantly expressed in certain cell types, such as the immune-related cells and rapidly regenerating cells. Recent studies have demonstrated that loss of cathepsin E in mice impairs their normal immune responses. In antigen-presenting cells (APC) such as macrophages, dendritic cells, and microglia, cathepsin E is localized mainly in the endosomal component and regulates the nature and functions of these cells. Deficiency of cathepsin E in macrophages induces a novel form of lysosomal storage disorder manifesting the accumulation of major lysosomal membrane glycoproteins such as LAMP-1 and LAMP-2 and elevated lysosomal pH. Such alterations in these cells are linked to abnormal intracellular trafficking of secretory and cell surface proteins. In fact, cathepsin E deficiency leads to increased secretion of a variety of soluble lysosomal enzymes including cathepsins and glycosidases, into the culture medium. By contrast, the expression and localization of cell surface proteins including Toll-like receptors, chemotactic receptors, and cell-adhesion receptors, as well as LAMPs, is significantly decreased by cathepsin E deficiency. While these alterations are not observed with cathepsin E-deficient (CatE-/-) dendritic cells, the cell surface expression and localization of the costimulatory molecules CD86, CD80, and CD40 were significantly increased in these cells, indicating that cathepsin E differentially regulates the nature and function of these two APC. This review focuses on the emerging roles of cathepsin E in proper intracellular trafficking of both secretory and cell surface proteins in APC. © 2012 Japanese Association for Oral Biology.

    DOI: 10.1016/j.job.2011.10.001

    Scopus

    researchmap

  • The role of cathepsin E in terminal differentiation of keratinocytes. Reviewed

    Kawakubo T, Yasukochi A, Okamoto K, Okamoto Y, Nakamura S, Yamamoto K

    Biological chemistry   392 ( 6 )   571 - 585   2011.4

  • Cathepsin E-deficient mice show increased susceptibility to bacterial infection associated with the decreased expression of multiple cell surface Toll-like receptors. Reviewed

    Tsukuba T, Yamamoto S, Yanagawa M, Okamoto K, Okamoto Y, Nakayama KI, Kadowaki T, Yamamoto K

    Journal of biochemistry   140 ( 1 )   57 - 66   2006.7

  • Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E1 Reviewed

    Jian Liu, Takayuki Tsukuba, Kuniaki Okamoto, Masamichi Ohishi, Kenji Yamamoto

    Journal of Biochemistry   132 ( 3 )   493 - 499   2002

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif. Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis. Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells. The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions. However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively. The Km values of these two mutants were similar to those of the wild-type enzyme, but their kcat values were greatly decreased. The Ki values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same. The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values. These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product. © 2002 Japanese Biochemical Society.

    DOI: 10.1093/oxfordjournals.jbchem.a003247

    Scopus

    PubMed

    researchmap

  • Characterization of phagosomal subpopulations along endocytic routes in osteoclasts and macrophages. Reviewed

    Sakai E, Miyamoto H, Okamoto K, Kato Y, Yamamoto K, Sakai H

    J Biochem.   130 ( 6 )   823 - 831   2001

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    researchmap

  • Design and synthesis of sensitive fluorogenic substrates specific for Lys-gingipain Reviewed

    Naoko Abe, Atsuyo Baba, Tomoko Kadowaki, Kuniaki Okamoto, Shinji Okazaki, Tetsuji Asao, Kenji Yamamoto

    Journal of Biochemistry   128 ( 5 )   877 - 881   2000

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press  

    Lys-gingipain (Kgp) is a major cysteine proteinase produced by the oral anaerobic bacterium Porphyromonas gingivalis, and has been implicated as a major pathogen in the development and progression of advanced adult periodontitis. This enzyme is believed to act as a major virulence factor of the disease, yet there exist no convenient and sensitive substrates for analyzing its biological activity. For a better understanding of the importance of this enzyme in the organism, there is an urgent need for specific substrates. Here we designed and synthesized two peptide 4-methyl-coumaryl-7-amides (MCA), carbobenzoxy (Z)-His-Glu-Lys-MCA, and Z-Glu-Lys-MCA, and tested their possible use as sensitive substrates for Kgp with limited specificity. Both substrates exhibited greater k(cat)/K(m) values than the best known Kgp substrates described so far. Both substrates were resistant to Arg-gingipain, another pathogenic cysteine proteinase from P. gingivalis, as well as trypsin and cathepsins B, L, and H. The levels of Kgp in various microorganisms and human cells were determined with Z-His-Glu-Lys-MCA. Little or no Kgp-like activity was detected in either other microorganisms or human cells tested. These results indicate that the present substrates are a valuable and fast tool for routine assays and for mechanistic studies on Kgp.

    DOI: 10.1093/oxfordjournals.jbchem.a022826

    Scopus

    researchmap

  • Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant Reviewed

    Yoshiyuki Yasuda, Shinobu Ikeda, Hideaki Sakai, Takayuki Tsukuba, Kuniaki Okamoto, Kazuhisa Nishishita, Akifumi Akamine, Yuzo Kato, Kenji Yamamoto

    European Journal of Biochemistry   266 ( 2 )   383 - 391   1999.12

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N- glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N- glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex- type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high- mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its- proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.

    DOI: 10.1046/j.1432-1327.1999.00863.x

    Scopus

    PubMed

    researchmap

  • Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis : construction of mutants with rgpA, rgpB, kgp, and hagA.

    Shi Y, Ratnayake DB, Okamoto K, Abe N, Yamamoto K, Nakayama K

    Journal of Biological Chemistry   274 ( 25 )   17955 - 17960   1999

  • Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-Gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease Reviewed

    Naoko Abe, Tomoko Kadowaki, Kuniaki Okamoto, Koji Nakayama, Masamichi Ohishi, Kenji Yamamoto

    Journal of Biochemistry   123 ( 2 )   305 - 312   1998

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press  

    The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed 'Lys-gingipain,' was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of M(r) = 51,000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and α1-antichymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.

    DOI: 10.1093/oxfordjournals.jbchem.a021937

    Scopus

    PubMed

    researchmap

  • Arg-gingipain acts as a major processing enzyme for various cell surface proteins in Porphyromonas gingivalis. Reviewed

    Kadowaki T, Nakayama K, Yoshimura F, Okamoto K, Abe N, Yamamoto K

    J. Biol. Chem.   273   29072 - 29076   1998

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1074/jbc.273.44.29072

    researchmap

  • Involvement of a Lysine-specific cysteine Proteinase in Hemoglobin Adsorption and Heme Accumulation by Porphyromonas gingivalis. Reviewed

    Okamoto K, Nakayama K, Kadowaki T, Abe N, Ratnayake D.B, Yamamoto K

    J. Biol. Chem.   273   21225 - 21231   1998

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1074/jbc.273.33.21225

    researchmap

  • Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-Gingipain) in porphyromonas gingivalis: Structural relationship with the arginine-specific cysteine proteinase (Arg-Gingipain) Reviewed

    Kuniaki Okamoto, Tomoko Kadowaki, Koji Nakayama, Kenji Yamamoto

    Journal of Biochemistry   120 ( 2 )   398 - 406   1996

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press  

    Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the I(GP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.

    DOI: 10.1093/oxfordjournals.jbchem.a021426

    Scopus

    researchmap

  • Structure and function of a novel arginine-specific cysteine proteinase (argingipain) as a major periodontal pathogenic factor from Porphyromonas gingivalis Reviewed

    Kenji Yamamoto, Tomoko Kadowaki, Kuniaki Okamoto, Masahiro Yoneda, Koji Nakayama, Yoshio Misumi, Yukio Ikehara

    Advances in Experimental Medicine and Biology   389   33 - 42   1996

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    Scopus

    PubMed

    researchmap

  • Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis. Reviewed

    Okamoto K, Misumi Y, Kadowaki T, Yoneda M, Yamamoto K

    Arch. Biochem. Biophys.   316   917 - 925   1995

     More details

    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1006/abbi.1995.1123

    researchmap

  • Construction and characterization of arginine-specific cysteine proteinase(Arg-ginigpain)-deficient mutants of Porphyromonas gingivalis : evidence for singificant contribution of Arg-gingipain to virulence.

    Nakayama K, Kadowaki T, Okamoto K, Yamamoto K

    Journal of Biological Chemistry   270 ( 40 )   23619 - 23626   1995

▼display all

Books

  • 第6版現代歯科薬理学

    岡元 邦彰( Role: Contributor ,  脂質異常治療薬 痛風治療薬)

    医歯薬出版株式会社  2017 

     More details

  • 第5版現代歯科薬理学

    岡元 邦彰( Role: Contributor ,  血液および造血臓器に作用する薬物 脂質異常治療薬 痛風治療薬)

    医歯薬出版株式会社  2012 

     More details

  • 「第4版現代歯科薬理学」

    岡元 邦彰( Role: Contributor ,  止血に用いられる薬物 血液および造血臓器に作用する薬物)

    医歯薬出版株式会社  2005 

     More details

  • Arg-gingipain and Lys-gingipain: a novel class of cysteine proteinases.

    Yamamoto K, Kadowaki T, Okamoto K( Role: Joint author)

    Birkhäusen Verlag  1999 

     More details

  • Structural characterization of pathogenic cysteine proteinase from the oral bacterium Porphysromonas gingivalis

    IOS Press  1997 

     More details

  • Possible roles of N-linked oligosaccharides on cathepsin E

    IOS Press  1997 

     More details

  • Possible roles of N-linked oligosaccharides on cathepsin E

    IOS Press  1997 

     More details

  • Structural characterization of pathogenic cysteine proteinase from the oral bacterium Porphysromonas gingivalis

    IOS Press  1997 

     More details

  • Biosynthesis and trafficking of cathepsin E

    IOS Press  1997 

     More details

  • Biosynthesis and trafficking of cathepsin E

    IOS Press  1997 

     More details

  • Biosynthesis and trafficking of cathepsin E

    IOS Press  1997 

     More details

  • Structural characterization of pathogenic cysteine proteinase from the oral bacterium Porphysromonas gingivalis

    IOS Press  1997 

     More details

  • Possible roles of N-linked oligosaccharides on cathepsin E

    IOS Press  1997 

     More details

▼display all

MISC

  • ニコチンは口腔扁平上皮癌細胞のセツキシマブ耐性を促進する

    清水 理恵子, 伊原木 聰一郎, 江口 傑徳, 奥井 達雄, 高畠 清文, 河合 穂高, 小野 喜章, 岡元 邦彰, 長塚 仁, 佐々木 朗

    岡山歯学会雑誌   37 ( 2 )   80 - 81   2018.12

     More details

    Language:Japanese   Publisher:岡山歯学会  

    researchmap

  • 口腔扁平上皮癌診断・治療における分子シャペロンHSP90含有細胞外小胞の可能性

    小野 喜章, 江口 傑徳, 十川 千春, 奥舎 有加, 河合 穂高, 中野 敬介, 佐々木 朗, 岡元 邦彰, 小崎 健一

    Journal of Oral Biosciences Supplement   2018   333 - 333   2018.9

     More details

    Language:Japanese   Publisher:(一社)歯科基礎医学会  

    researchmap

  • 核内およびエクソソーム中に存在するMMPs/PEXの癌進展における役割とその抑制

    奥舎 有加, 江口 傑徳, 十川 千春, 小野 喜章, 奥井 達雄, 中野 敬介, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2018   150 - 150   2018.9

     More details

    Language:Japanese   Publisher:(一社)歯科基礎医学会  

    researchmap

  • 癌の治療抵抗性と転移におけるHSP90およびMMP3の役割

    江口 傑徳, 小野 喜章, 奥舎 有加, 十川 千春, 内部 健太, 中野 敬介, 奥井 達雄, 滝川 正春, 岡元 邦彰, カルダーウッド・スチュアート

    Journal of Oral Biosciences Supplement   2018   142 - 142   2018.9

     More details

    Language:Japanese   Publisher:(一社)歯科基礎医学会  

    researchmap

  • New functions of lysosomes in bone cells

    Takayuki Tsukuba, Eiko Sakai, Kazuhisa Nishishita, Tomoko Kadowaki, Kuniaki Okamoto

    JOURNAL OF ORAL BIOSCIENCES   59 ( 2 )   92 - 95   2017.5

     More details

    Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:ELSEVIER SCIENCE BV  

    Background: Lysosomes are intracellular acidic organelles that contain approximately 50 hydrolases and 25 species of integral membrane proteins. Although lysosome-like specific compartments, termed lysosome-related organelles (LROs), are found in osteoclasts, their functions in these cells and lysosomal functions in osteoblasts remain to be elucidated.Highlight: Recently, we found that expression of RAB27A is markedly increased during osteoclastic differentiation. RAB27A deficiency causes multinucleation and giant cell formation, characterized by abnormal transport of cell surface receptors and LROs into osteoclasts. Furthermore, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. Overexpression of TFEB in preosteoblastic MC3T3-E1 cells enhances osteoblastic differentiation via decreased expression of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). These results indicate that the expression of ATF4 and CHOP is essential for differentiation into osteoblasts.Conclusion: RAB27A participates in bone resorption by LROs in osteoclasts. In addition, lysosomal biogenesis modulated by TFEB is necessary for osteoblastic differentiation. (C) 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.job.2017.01.004

    Web of Science

    researchmap

  • 新規高分子量Gタンパク質Rab44はCa2+流入及びNFATc1経路を介して破骨細胞分化を負に制御する

    山口優, 坂井詠子, 岡元邦彰, 鍜治屋浩, 岡部幸司, 門脇知子, 筑波隆幸

    日本生化学会大会(Web)   90th   2017

  • 破骨細胞分化を負に制御する新規Kelchタンパク質の同定と機能解析

    楢原峻, 楢原峻, 坂井詠子, 山口優, 楢原春菜, 門脇知子, 岡元邦彰, 朝比奈泉, 筑波隆幸

    日本生化学会大会(Web)   90th   2017

  • 破骨細胞分化を制御する新規Rabタンパク質の同定と解析

    山口優, 坂井詠子, 岡元邦彰, 門脇知子, 筑波隆幸

    日本病態プロテアーゼ学会学術集会プログラム抄録集   22nd   2017

  • コバルトプロトポルフィリンの破骨細胞分化および活性化に対する影響

    八島由佳, 八島由佳, 岡元邦彰, 坂井詠子, 西下一久, 筑波隆幸

    Journal of Oral Biosciences Supplement (Web)   2014   ROMBUNNO.P2‐33 (WEB ONLY)   2014

     More details

    Language:Japanese  

    J-GLOBAL

    researchmap

  • Chemical constituent of Sanguisorba officinalis inhibits osteoclastogenesis

    Eiko Sakai, Mayumi Iwatake, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   124   232P - 232P   2014

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Up-regulation mechanisms of endosomal/lysosomal proteins during osteoblast differentiation

    Erika Yoneshima, Kuniaki Okamoto, Eiko Sakai, Noriaki Yoshida, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   121   170P - 170P   2013

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Cafestol prevents osteoclastogenesis via impairment of NFATc1 expression and blocking of Erk phosphorylation

    Yutaka Fukuma, Eiko Sakai, Megumi Sugawara, Erika Yoneshima, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   121   249P - 249P   2013

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Fisetinl prevents osteoclastogenesis via impairment of NFATc1 expression and blocking of Erk phosphorylation

    Megumi Sugawara, Eiko Sakai, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Noriaki Yoshida, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118   164P - 164P   2012

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Identification of two transcrips and in vivo promoter analysis for cathepsin E

    Kuniaki Okamoto, Yoshiko Okamoto, Eiko Sakai, Kazuhisa Nishishita, Kenji Yamamoto, Takayuki Tstikuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118   259P - 259P   2012

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Accumulation of NADPH oxidase (NOX2) and increased oxidative stress in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Kenji Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118   256P - 256P   2012

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Nrf2 activators inhibit osteoclastogenesis induced by receptor activator of NF-kappa B ligand

    Reiko Fumimoto, Hiroshi Sakamoto, Yu Yamaguchi, Eiko Sakai, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   115   219P - 219P   2011

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Receptor activator of NF-kappa B ligand dependent heme oxygenase-1 suppression is required for high mobility group box 1 release, caspase-3 activation, and subsequent osteoclastogenesis

    Eiko Sakai, Megumi Shimada, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   115   219P - 219P   2011

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Transcription factor Sp1 regulates the expression of the murine cathepsin E gene in gastric adenosarcoma cells

    Kuniaki Okamoto, Yoshiko Okamoto, Tomoyo Kawakubo, Kenji Yamamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   112   216P - 216P   2010

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Autophagy impairment associated with increased aberrant mitochondria and oxidative stress in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Michiyo Yanagawa, Tomoko Kadowaki, Yoshiko Okamoto, Kuniaki Okamoto, Kenji Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   112   216P - 216P   2010

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells

    Y. Yasuda, E. Ohtomo, T. Tsukuba, K. Okamoto, T. Saito

    INTERNATIONAL ENDODONTIC JOURNAL   42 ( 10 )   940 - 946   2009.10

     More details

    Language:English   Publisher:WILEY-BLACKWELL PUBLISHING, INC  

    P>Aim
    To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells.
    Methodology
    Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test.
    Results
    The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively.
    Conclusions
    These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

    DOI: 10.1111/j.1365-2591.2009.01598.x

    Web of Science

    researchmap

  • Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells

    Y. Yasuda, E. Ohtomo, T. Tsukuba, K. Okamoto, T. Saito

    INTERNATIONAL ENDODONTIC JOURNAL   42 ( 10 )   940 - 946   2009.10

     More details

    Language:English   Publisher:WILEY-BLACKWELL PUBLISHING, INC  

    P>Aim
    To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells.
    Methodology
    Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test.
    Results
    The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively.
    Conclusions
    These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

    DOI: 10.1111/j.1365-2591.2009.01598.x

    Web of Science

    researchmap

  • Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Michiyo Yanagawa, Kuniaki Okamoto, Yoshiko Okamoto, Yoshiyuki Yasuda, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOCHEMISTRY   145 ( 5 )   565 - 573   2009.5

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE(/)) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE(/)macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE(/)macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin (2)) and CD 29 (integrin (1)) in CatE(/) macrophages were significantly decreased, thereby reducing cell attachment of CatE(/) macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE(/)macrophages.

    DOI: 10.1093/jb/mvp016

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Michiyo Yanagawa, Kuniaki Okamoto, Yoshiko Okamoto, Yoshiyuki Yasuda, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOCHEMISTRY   145 ( 5 )   565 - 573   2009.5

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE(/)) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE(/)macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE(/)macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin (2)) and CD 29 (integrin (1)) in CatE(/) macrophages were significantly decreased, thereby reducing cell attachment of CatE(/) macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE(/)macrophages.

    DOI: 10.1093/jb/mvp016

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • カテプシンE欠損マクロファージにおけるオートファジーの低下とそれに伴うミトコンドリア機能異常と酸化ストレスの上昇

    筑波隆幸, 門脇知子, 坂井詠子, 岡元邦彰, 山本健二

    Journal of Oral Biosciences   51 ( Supplement )   2009

  • Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system

    Yutaka Ishida, JinPing Hu, Eiko Sakai, Tomoko Kadowaki, Kenji Yamamoto, Takayuki Tsukuba, Yuzo Kato, Koji Nakayama, Kuniaki Okamoto

    ARCHIVES OF ORAL BIOLOGY   53 ( 6 )   538 - 544   2008.6

     More details

    Language:English   Publisher:PERGAMON-ELSEVIER SCIENCE LTD  

    Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.archoralbio.2008.01.004

    Web of Science

    researchmap

  • Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system

    Yutaka Ishida, JinPing Hu, Eiko Sakai, Tomoko Kadowaki, Kenji Yamamoto, Takayuki Tsukuba, Yuzo Kato, Koji Nakayama, Kuniaki Okamoto

    ARCHIVES OF ORAL BIOLOGY   53 ( 6 )   538 - 544   2008.6

     More details

    Language:English   Publisher:PERGAMON-ELSEVIER SCIENCE LTD  

    Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.archoralbio.2008.01.004

    Web of Science

    researchmap

  • Dentin phosphophoryn promotes cellular migration of human dental pulp cells

    Yoshiyuki Yasuda, Masanobu Izumikawa, Kuniaki Okamoto, Takayuki Tsukuba, Takashi Saito

    JOURNAL OF ENDODONTICS   34 ( 5 )   575 - 578   2008.5

     More details

    Language:English   Publisher:ELSEVIER SCIENCE INC  

    Dentin phosphophoryn (DPP) is a dentin sialophosphoprotein gene product that has an RGD motif and repeat sequences of aspartic acid and phosphoserine. To date, the function of DPP in the early stage of reparative dentin formation still remains unclear. The objective of this study was to evaluate the effects of DPP on pulp cell migration and proliferation. DPP promoted cell migration in a concentration-dependent manner, thus increasing it by about 3-fold at 1000 ng/mL compared with the control, but it had no effect on cell proliferation. Dephosphorylated DPP also promoted cell migration, similarly to DPP. However, cell migration was significantly suppressed by the addition of alpha v beta 3 integrin antibody to the medium. Furthermore, porcine DPP-derived RGD peptide, but not its mutant RAD peptide, significantly promoted cell migration. These results indicated that the RGD motif of DPP plays an important role in the migration of human dental pulp cells.

    DOI: 10.1016/j.joen.2008.02.018

    Web of Science

    researchmap

  • Association of cathepsin E deficiency with the increased territorial aggressive response of mice

    Naoki Shigematsu, Takaichi Fukuda, Tsuneyuki Yamamoto, Tsuyoshi Nishioku, Taku Yamaguchi, Masaru Himeno, Keiichi I. Nakayama, Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Shun Higuchi, Kenji Yamamoto

    JOURNAL OF NEUROCHEMISTRY   105 ( 4 )   1394 - 1404   2008.5

     More details

    Language:English   Publisher:WILEY  

    Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE-/-) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE-/- mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE-/- mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE-/- mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.

    DOI: 10.1111/j.1471-4159.2008.05242.x

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Dentin phosphophoryn promotes cellular migration of human dental pulp cells

    Yoshiyuki Yasuda, Masanobu Izumikawa, Kuniaki Okamoto, Takayuki Tsukuba, Takashi Saito

    JOURNAL OF ENDODONTICS   34 ( 5 )   575 - 578   2008.5

     More details

    Language:English   Publisher:ELSEVIER SCIENCE INC  

    Dentin phosphophoryn (DPP) is a dentin sialophosphoprotein gene product that has an RGD motif and repeat sequences of aspartic acid and phosphoserine. To date, the function of DPP in the early stage of reparative dentin formation still remains unclear. The objective of this study was to evaluate the effects of DPP on pulp cell migration and proliferation. DPP promoted cell migration in a concentration-dependent manner, thus increasing it by about 3-fold at 1000 ng/mL compared with the control, but it had no effect on cell proliferation. Dephosphorylated DPP also promoted cell migration, similarly to DPP. However, cell migration was significantly suppressed by the addition of alpha v beta 3 integrin antibody to the medium. Furthermore, porcine DPP-derived RGD peptide, but not its mutant RAD peptide, significantly promoted cell migration. These results indicated that the RGD motif of DPP plays an important role in the migration of human dental pulp cells.

    DOI: 10.1016/j.joen.2008.02.018

    Web of Science

    researchmap

  • Association of cathepsin E deficiency with the increased territorial aggressive response of mice

    Naoki Shigematsu, Takaichi Fukuda, Tsuneyuki Yamamoto, Tsuyoshi Nishioku, Taku Yamaguchi, Masaru Himeno, Keiichi I. Nakayama, Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Shun Higuchi, Kenji Yamamoto

    JOURNAL OF NEUROCHEMISTRY   105 ( 4 )   1394 - 1404   2008.5

     More details

    Language:English   Publisher:WILEY  

    Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE-/-) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE-/- mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE-/- mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE-/- mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.

    DOI: 10.1111/j.1471-4159.2008.05242.x

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Berberine inhibits RANKL-induced osteoclast formation and survival through suppressing the NF-kappa B and Akt pathways

    Jin-Ping Hu, Kazuhisa Nishishita, Eiko Sakai, Hajime Yoshida, Yuzo Kato, Takayuki Tsukuba, Kuniaki Okamoto

    EUROPEAN JOURNAL OF PHARMACOLOGY   580 ( 1-2 )   70 - 79   2008.2

     More details

    Language:English   Publisher:ELSEVIER SCIENCE BV  

    Berberine, an isoquinoline alkaloid isolated from several medicinal plants, has been reported to possess anti-bacterial, anti-inflammatory and antitumor properties. Although berberine also inhibits osteoclastogenesis and bone resorption, the molecular machinery for its inhibitory effects remains unknown. This study focused on the suppressive effects of berberine on receptor activator of nuclear factor kappa B (NF-kappa B) ligand (RANKL)induced osteoclastogenesis and survival. Berberine inhibited RANKL-mediated osteoclast fort-nation and survival while having no cytotoxic effects on bone marrow macrophages or osteoblastic cells. Berberine attenuated RANKL-induced activation of NF-kappa B through inhibiting phosphorylation at the activation loop of I kappa B alpha kinase, phosphorylation and degradation of I kappa B alpha, and NF-kappa B p65 nuclear translocation. RANKL-induced Akt phosphorylation was strongly inhibited by berberine; however, neither monocyte/macrophage-colony stimulating factor (M-CSF)-induced nor insulin-induced Akt activation was inhibited by the drug. Under M-CSF- and RANKL-deprived condition, berberine increased the active form of caspase-3 in osteoclasts. By contrast, berberine did not potentiate the activation of caspase-3 in M-CSF-deprived bone marrow macrophages. These findings indicate that berberine inhibits osteoclast formation and survival through suppression of NF-kappa B and Akt activation and that both pathways in the osteoclast lineage are highly sensitive to berberine treatment. (C) 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.ejphar.2007.11.013

    Web of Science

    researchmap

  • Berberine inhibits RANKL-induced osteoclast formation and survival through suppressing the NF-kappa B and Akt pathways

    Jin-Ping Hu, Kazuhisa Nishishita, Eiko Sakai, Hajime Yoshida, Yuzo Kato, Takayuki Tsukuba, Kuniaki Okamoto

    EUROPEAN JOURNAL OF PHARMACOLOGY   580 ( 1-2 )   70 - 79   2008.2

     More details

    Language:English   Publisher:ELSEVIER SCIENCE BV  

    Berberine, an isoquinoline alkaloid isolated from several medicinal plants, has been reported to possess anti-bacterial, anti-inflammatory and antitumor properties. Although berberine also inhibits osteoclastogenesis and bone resorption, the molecular machinery for its inhibitory effects remains unknown. This study focused on the suppressive effects of berberine on receptor activator of nuclear factor kappa B (NF-kappa B) ligand (RANKL)induced osteoclastogenesis and survival. Berberine inhibited RANKL-mediated osteoclast fort-nation and survival while having no cytotoxic effects on bone marrow macrophages or osteoblastic cells. Berberine attenuated RANKL-induced activation of NF-kappa B through inhibiting phosphorylation at the activation loop of I kappa B alpha kinase, phosphorylation and degradation of I kappa B alpha, and NF-kappa B p65 nuclear translocation. RANKL-induced Akt phosphorylation was strongly inhibited by berberine; however, neither monocyte/macrophage-colony stimulating factor (M-CSF)-induced nor insulin-induced Akt activation was inhibited by the drug. Under M-CSF- and RANKL-deprived condition, berberine increased the active form of caspase-3 in osteoclasts. By contrast, berberine did not potentiate the activation of caspase-3 in M-CSF-deprived bone marrow macrophages. These findings indicate that berberine inhibits osteoclast formation and survival through suppression of NF-kappa B and Akt activation and that both pathways in the osteoclast lineage are highly sensitive to berberine treatment. (C) 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.ejphar.2007.11.013

    Web of Science

    researchmap

  • カテプシンE欠損はオートファジーの低下とそれに伴うミトコンドリア機能異常と酸化ストレスを引き起こす。

    筑波隆幸, 柳川三千代, 門脇知子, 岡本美子, 岡元邦彰, 中山敬一, 山本健二

    生化学   2008

  • カテプシンE欠損マウスにおける高脂血症および脂肪肝の誘導

    門脇知子, 岡元邦彰, 山本健二, 筑波隆幸

    Journal of Oral Biosciences   50 ( Supplement )   2008

  • カテプシンE欠損による遊走能および細胞接着能の低下

    筑波隆幸, 柳川三千代, 岡元邦彰, 門脇知子, 山本健二

    Journal of Oral Biosciences   50 ( Supplement )   2008

  • Cathepsin E prevents tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface

    Tomoyo Kawakubo, Kuniaki Okamoto, Jun-Ichi Lwata, Masashi Shin, Yoshiko Okamoto, Atsushi Yasukochi, Keiichi I. Nakayama, Tomoko Kadowaki, Takayuki Tsukuba, Kenji Yamamoto

    CANCER RESEARCH   67 ( 22 )   10869 - 10878   2007.11

     More details

    Language:English   Publisher:AMER ASSOC CANCER RESEARCH  

    The aspartic proteinase cathepsin E is expressed predominantly in cells of the immune system and highly secreted by activated phagocytes, and deficiency of cathepsin E in mice results in a phenotype affecting immune responses. However, because physiologic substrates for cathepsin E have not yet been identified, the relevance of these observations to the physiologic functions of this protein remains speculative. Here, we show that cathepsin E specifically induces growth arrest and apoptosis in human prostate carcinoma tumor cell lines without affecting normal cells by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. The antitumor activity of cathepsin E was corroborated by in vivo studies with mice bearing human and mouse tumor transplants. Administration of purified cathepsin E into human tumor xenografts in nude mice dose-dependently induced apoptosis in the tumor cells to inhibit tumor growth. The growth, viability, and metastasis of mouse B16 melanoma cells were also more profound in cathepsin E-deficient mice compared with those in the syngeneic wild-type and transgenic mice overexpressing cathepsin E. Taken together, the number of apoptotic tumor cells, as well as tumor-infiltrating activated macrophages, was apparently reduced in cathepsin E-deficient mice compared with those in the other two groups, implying the positive correlation of endogenous cathepsin E levels with the extent of tumor suppression in vivo. These results thus indicate thatcathepsinEplaysasubstantialroleinhostdefense against tumor cells through TRAIL-dependent apoptosis and/or tumor-associated macrophage-mediated cytotoxicity.

    DOI: 10.1158/0008-5472.CAN-07-2048

    Web of Science

    researchmap

  • Cathepsin E prevents tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface

    Tomoyo Kawakubo, Kuniaki Okamoto, Jun-Ichi Lwata, Masashi Shin, Yoshiko Okamoto, Atsushi Yasukochi, Keiichi I. Nakayama, Tomoko Kadowaki, Takayuki Tsukuba, Kenji Yamamoto

    CANCER RESEARCH   67 ( 22 )   10869 - 10878   2007.11

     More details

    Language:English   Publisher:AMER ASSOC CANCER RESEARCH  

    The aspartic proteinase cathepsin E is expressed predominantly in cells of the immune system and highly secreted by activated phagocytes, and deficiency of cathepsin E in mice results in a phenotype affecting immune responses. However, because physiologic substrates for cathepsin E have not yet been identified, the relevance of these observations to the physiologic functions of this protein remains speculative. Here, we show that cathepsin E specifically induces growth arrest and apoptosis in human prostate carcinoma tumor cell lines without affecting normal cells by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. The antitumor activity of cathepsin E was corroborated by in vivo studies with mice bearing human and mouse tumor transplants. Administration of purified cathepsin E into human tumor xenografts in nude mice dose-dependently induced apoptosis in the tumor cells to inhibit tumor growth. The growth, viability, and metastasis of mouse B16 melanoma cells were also more profound in cathepsin E-deficient mice compared with those in the syngeneic wild-type and transgenic mice overexpressing cathepsin E. Taken together, the number of apoptotic tumor cells, as well as tumor-infiltrating activated macrophages, was apparently reduced in cathepsin E-deficient mice compared with those in the other two groups, implying the positive correlation of endogenous cathepsin E levels with the extent of tumor suppression in vivo. These results thus indicate thatcathepsinEplaysasubstantialroleinhostdefense against tumor cells through TRAIL-dependent apoptosis and/or tumor-associated macrophage-mediated cytotoxicity.

    DOI: 10.1158/0008-5472.CAN-07-2048

    Web of Science

    researchmap

  • Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide. International journal

    Eiko Sakai, Mariko Naito, Keiko Sato, Hitoshi Hotokezaka, Tomoko Kadowaki, Arihide Kamaguchi, Kenji Yamamoto, Kuniaki Okamoto, Koji Nakayama

    Journal of bacteriology   189 ( 11 )   3977 - 86   2007.6

     More details

    Language:English  

    Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

    DOI: 10.1128/JB.01691-06

    PubMed

    CiNii Article

    researchmap

  • Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide. International journal

    Eiko Sakai, Mariko Naito, Keiko Sato, Hitoshi Hotokezaka, Tomoko Kadowaki, Arihide Kamaguchi, Kenji Yamamoto, Kuniaki Okamoto, Koji Nakayama

    Journal of bacteriology   189 ( 11 )   3977 - 86   2007.6

     More details

    Language:English  

    Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

    DOI: 10.1128/JB.01691-06

    PubMed

    CiNii Article

    researchmap

  • Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

    Michiyo Yanagawa, Takayuki Tsukuba, Tsuyoshi Nishioku, Yoshiko Okamoto, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 3 )   1851 - 1862   2007.1

     More details

    Language:English   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+ -ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.

    DOI: 10.1074/jbc.M604143200

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

    Michiyo Yanagawa, Takayuki Tsukuba, Tsuyoshi Nishioku, Yoshiko Okamoto, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 3 )   1851 - 1862   2007.1

     More details

    Language:English   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+ -ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.

    DOI: 10.1074/jbc.M604143200

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • カテプシンE欠損はマクロファージの膜蛋白質輸送異常とミトコンドリア機能異常を起こす

    筑波隆幸, 柳川三千代, 岡元邦彰, 岡本美子, 門脇知子, 山本健二

    生化学   2007

  • Cathepsin E mediates membrane trafficking in macrophages

    Takayuki Tsukuba, Shinya Yamamoto, Michiyo Yanagawa, Yoshiko Okamoto, Kuniaki Okamoto, Kenji Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   103   214P - 214P   2007

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • カテプシンE欠損によるミトコンドリア機能異常と酸化ストレス亢進

    筑波隆幸, 柳川三千代, 柳川三千代, 岡元邦彰, 門脇知子, 山本健二

    Journal of Oral Biosciences   49 ( Supplement )   2007

  • P.gingivalis感染とインスリン抵抗性に関する実験的解析

    門脇知子, 瀧井良祐, 筑波隆幸, 岡元邦彰, 山本健二

    Journal of Oral Biosciences   49 ( Supplement )   2007

  • Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation. International journal

    Hajime Yoshida, Kuniaki Okamoto, Tsutomu Iwamoto, Eiko Sakai, Kazuhiro Kanaoka, Jin-Ping Hu, Mitsue Shibata, Hitoshi Hotokezaka, Kazuhisa Nishishita, Akio Mizuno, Yuzo Kato

    Journal of biochemistry   139 ( 3 )   583 - 90   2006.3

     More details

    Language:English  

    Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.

    DOI: 10.1093/jb/mvj066

    PubMed

    CiNii Article

    researchmap

  • Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation. International journal

    Hajime Yoshida, Kuniaki Okamoto, Tsutomu Iwamoto, Eiko Sakai, Kazuhiro Kanaoka, Jin-Ping Hu, Mitsue Shibata, Hitoshi Hotokezaka, Kazuhisa Nishishita, Akio Mizuno, Yuzo Kato

    Journal of biochemistry   139 ( 3 )   583 - 90   2006.3

     More details

    Language:English  

    Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.

    DOI: 10.1093/jb/mvj066

    PubMed

    CiNii Article

    researchmap

  • Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

    T Tsukuba, S Ikeda, K Okamoto, Y Yasuda, E Sakai, T Kadowaki, H Sakai, K Yamamoto

    FEBS JOURNAL   273 ( 1 )   219 - 229   2006.1

     More details

    Language:English   Publisher:WILEY-BLACKWELL  

    To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.

    DOI: 10.1111/j.1742-4658.2005.05062.x

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

    T Tsukuba, S Ikeda, K Okamoto, Y Yasuda, E Sakai, T Kadowaki, H Sakai, K Yamamoto

    FEBS JOURNAL   273 ( 1 )   219 - 229   2006.1

     More details

    Language:English   Publisher:WILEY-BLACKWELL  

    To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.

    DOI: 10.1111/j.1742-4658.2005.05062.x

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2- and TLR4-independent signaling

    M Kishimoto, A Yoshimura, M Naito, K Okamoto, K Yamamoto, DT Golenbock, Y Hara, K Nakayama

    MICROBIOLOGY AND IMMUNOLOGY   50 ( 4 )   315 - 325   2006

     More details

    Language:English   Publisher:CENTER ACADEMIC PUBL JAPAN  

    Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P gingivalis cells and activation of nuclear factor-kappa B in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P gingivalis, they responded to gingipain-deficient P gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P gingivalis but have inhibitory effects on TLR2- and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of R gingivalis that were able to induce TLR2- and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P gingivalis to escape from the innate immune system.

    Web of Science

    researchmap

  • Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2- and TLR4-independent signaling

    M Kishimoto, A Yoshimura, M Naito, K Okamoto, K Yamamoto, DT Golenbock, Y Hara, K Nakayama

    MICROBIOLOGY AND IMMUNOLOGY   50 ( 4 )   315 - 325   2006

     More details

    Language:English   Publisher:CENTER ACADEMIC PUBL JAPAN  

    Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P gingivalis cells and activation of nuclear factor-kappa B in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P gingivalis, they responded to gingipain-deficient P gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P gingivalis but have inhibitory effects on TLR2- and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of R gingivalis that were able to induce TLR2- and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P gingivalis to escape from the innate immune system.

    Web of Science

    researchmap

  • カテプシンE欠損マクロファージにおける膜タンパク質の輸送異常

    筑波隆幸, 山本晋也, 山本晋也, 柳川三千代, 柳川三千代, 岡元邦彰, 門脇知子, 山本健二

    Journal of Oral Biosciences   48 ( Supplement )   2006

  • P.gingivalis感染の生活習慣病増悪機序について

    門脇知子, 筑波隆幸, 瀧井良祐, 重松直樹, 岡元邦彰, 山本健二

    Journal of Oral Biosciences   48 ( Supplement )   2006

  • The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome

    Y Yasuda, T Tsukuba, K Okamoto, T Kadowaki, K Yamamoto

    JOURNAL OF BIOCHEMISTRY   138 ( 5 )   621 - 630   2005.11

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (k(cat)/K(m)) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.

    DOI: 10.1093/jb/mvi159

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome

    Y Yasuda, T Tsukuba, K Okamoto, T Kadowaki, K Yamamoto

    JOURNAL OF BIOCHEMISTRY   138 ( 5 )   621 - 630   2005.11

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (k(cat)/K(m)) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.

    DOI: 10.1093/jb/mvi159

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

    K Sato, E Sakai, PD Veith, M Shoji, Y Kikuchi, H Yukitake, N Ohara, M Naito, K Okamoto, EC Reynolds, K Nakayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 10 )   8668 - 8677   2005.3

     More details

    Language:English   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

    DOI: 10.1074/jbc.M413544200

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

    K Sato, E Sakai, PD Veith, M Shoji, Y Kikuchi, H Yukitake, N Ohara, M Naito, K Okamoto, EC Reynolds, K Nakayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 10 )   8668 - 8677   2005.3

     More details

    Language:English   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

    DOI: 10.1074/jbc.M413544200

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Up-regulation, Enhanced Maturation, and Secretion of Cathepsin E in Mouse Macrophages Treated with Interferon-γ or Lipopolysaccharide

    Michiyo Yanagawa, Takayuki Tsukuba, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Tomoko Kadowaki, Kenji Yamamoto

    journal of oral biosciences   48 ( 3 )   218 - 225   2005

     More details

    Language:English  

    Cathepsin E is an intracellular aspartic proteinase that is predominantly localized in the endosomal compartments of antigen presenting cells including macrophages. Here, we have investigated the expression of cathepsin E in mouse macrophages treated with interferon (IFN)-γ, lipopolysaccharide (LPS), interleukin (IL)-10, and IL-4. The mRNA levels of cathepsin E in macrophages stimulated with IFN-γ and LPS were increased, but conversely, that with IL-10 was decreased. However, upon stimulation with IFN-γ or LPS, the activity levels of cathepsin E in the activated cells were markedly decreased, but those in the culture media were increased. Immunoblot analysis revealed that procathepsin E in the cells was largely converted to the mature form in the cells upon stimulation with IFN-γ. In addition, the extracellular enzyme was reacted with antibodies to mature cathepsin E but not with antibodies for procathepsin E. By contrast, when macrophages were treated with IL-4, cathepsin E production was significantly decreased in both the cell and the medium. These results indicate that, upon stimulation with IFN-γ and LPS, cathepsin E is up-regulated in macrophages, which is accompanied by the enhanced maturation and secretion of the enzyme. Conversely, cathepsin E is down-regulated by treatment with IL-4 and IL-10. © 2006, Japanese Association for Oral Biology. All rights reserved.

    DOI: 10.2330/joralbiosci.48.218

    Scopus

    CiNii Article

    researchmap

  • Up-regulation, Enhanced Maturation, and Secretion of Cathepsin E in Mouse Macrophages Treated with Interferon-γ or Lipopolysaccharide

    Michiyo Yanagawa, Takayuki Tsukuba, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Tomoko Kadowaki, Kenji Yamamoto

    journal of oral biosciences   48 ( 3 )   218 - 225   2005

     More details

    Language:English  

    Cathepsin E is an intracellular aspartic proteinase that is predominantly localized in the endosomal compartments of antigen presenting cells including macrophages. Here, we have investigated the expression of cathepsin E in mouse macrophages treated with interferon (IFN)-γ, lipopolysaccharide (LPS), interleukin (IL)-10, and IL-4. The mRNA levels of cathepsin E in macrophages stimulated with IFN-γ and LPS were increased, but conversely, that with IL-10 was decreased. However, upon stimulation with IFN-γ or LPS, the activity levels of cathepsin E in the activated cells were markedly decreased, but those in the culture media were increased. Immunoblot analysis revealed that procathepsin E in the cells was largely converted to the mature form in the cells upon stimulation with IFN-γ. In addition, the extracellular enzyme was reacted with antibodies to mature cathepsin E but not with antibodies for procathepsin E. By contrast, when macrophages were treated with IL-4, cathepsin E production was significantly decreased in both the cell and the medium. These results indicate that, upon stimulation with IFN-γ and LPS, cathepsin E is up-regulated in macrophages, which is accompanied by the enhanced maturation and secretion of the enzyme. Conversely, cathepsin E is down-regulated by treatment with IL-4 and IL-10. © 2006, Japanese Association for Oral Biology. All rights reserved.

    DOI: 10.2330/joralbiosci.48.218

    Scopus

    CiNii Article

    researchmap

  • Porphyromonas gingivalisの産生するシステインプロテアーゼ(Kgp)の活性中心の決定

    岡元邦彰, HU P J, 坂井詠子, 西下一久, 門脇知子, 山本健二, 中山浩次, 加藤有三

    Journal of Oral Biosciences   47 ( Supplement )   2005

  • Cathepsin E is important for endosomal/lysosomal systems in macrophages against a bacterial infection

    T Tsukuba, M Yanagawa, Y Okamoto, K Okamoto, K Yanamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   97   78P - 78P   2005

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

    N Abe, A Baba, R Takii, K Nakayama, A Kamaguchi, Y Shibata, Y Abiko, K Okamoto, T Kadowaki, K Yamamoto

    BIOLOGICAL CHEMISTRY   385 ( 11 )   1041 - 1047   2004.11

     More details

    Language:English   Publisher:WALTER DE GRUYTER & CO  

    Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

    DOI: 10.1515/BC.2004.135

    Web of Science

    researchmap

  • Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

    N Abe, A Baba, R Takii, K Nakayama, A Kamaguchi, Y Shibata, Y Abiko, K Okamoto, T Kadowaki, K Yamamoto

    BIOLOGICAL CHEMISTRY   385 ( 11 )   1041 - 1047   2004.11

     More details

    Language:English   Publisher:WALTER DE GRUYTER & CO  

    Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

    DOI: 10.1515/BC.2004.135

    Web of Science

    researchmap

  • Laminin alpha 2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

    K Yuasa, S Fukumoto, Y Kamasaki, A Yamada, E Fukumoto, K Kanaoka, K Saito, H Harada, E Arikawa-Hirasawa, Y Miyagoe-Suzuki, S Takeda, K Okamoto, Y Kato, T Fujiwara

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 11 )   10286 - 10292   2004.3

     More details

    Language:English   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wildtype and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

    DOI: 10.1074/jbc.M310013200

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Laminin alpha 2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

    K Yuasa, S Fukumoto, Y Kamasaki, A Yamada, E Fukumoto, K Kanaoka, K Saito, H Harada, E Arikawa-Hirasawa, Y Miyagoe-Suzuki, S Takeda, K Okamoto, Y Kato, T Fujiwara

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 11 )   10286 - 10292   2004.3

     More details

    Language:English   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wildtype and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

    DOI: 10.1074/jbc.M310013200

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Association of cathepsin E deficiency with development of atopic dermatitis

    T Tsukuba, K Okamoto, Y Okamoto, M Yanagawa, K Kohmura, Y Yasuda, H Uchi, T Nakahara, M Furue, K Nakayama, T Kadowaki, K Yamamoto, KI Nakayama

    JOURNAL OF BIOCHEMISTRY   134 ( 6 )   893 - 902   2003.12

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal. aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4(+)/CD8(+) T cells, the strong polarization of naive T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1beta accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1beta were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1beta, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.

    DOI: 10.1093/jh/mvg216

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Association of cathepsin E deficiency with development of atopic dermatitis

    T Tsukuba, K Okamoto, Y Okamoto, M Yanagawa, K Kohmura, Y Yasuda, H Uchi, T Nakahara, M Furue, K Nakayama, T Kadowaki, K Yamamoto, KI Nakayama

    JOURNAL OF BIOCHEMISTRY   134 ( 6 )   893 - 902   2003.12

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal. aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4(+)/CD8(+) T cells, the strong polarization of naive T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1beta accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1beta were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1beta, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.

    DOI: 10.1093/jh/mvg216

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • The regulation of bone resorption in tooth formation and eruption processes in mouse alveolar crest devoid of cathepsin K

    M Okaji, H Sakai, E Sakai, M Shibata, F Hashimoto, Y Kobayashi, N Yoshida, K Okamoto, K Yamamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91 ( 4 )   285 - 294   2003.4

     More details

    Language:English   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.

    DOI: 10.1254/jphs.91.285

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • The regulation of bone resorption in tooth formation and eruption processes in mouse alveolar crest devoid of cathepsin K

    M Okaji, H Sakai, E Sakai, M Shibata, F Hashimoto, Y Kobayashi, N Yoshida, K Okamoto, K Yamamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91 ( 4 )   285 - 294   2003.4

     More details

    Language:English   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.

    DOI: 10.1254/jphs.91.285

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Disruption of structural and functional integrity of alpha(2)-macroglobulin by cathepsin E

    M Shibata, H Sakai, E Sakai, K Okamoto, K Nishishita, Y Yasuda, Y Kato, K Yamamoto

    EUROPEAN JOURNAL OF BIOCHEMISTRY   270 ( 6 )   1189 - 1198   2003.3

     More details

    Language:English   Publisher:BLACKWELL PUBLISHING LTD  

    alpha(2) -Macroglobulin (alpha2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha2M and biological targets, little is known about the nature of alpha2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha2M in the endolysosome system, we examined the capacity of alpha2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha2M in the endolysosome system.

    DOI: 10.1046/j.1432-1033.2003.03479.x

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Disruption of structural and functional integrity of alpha(2)-macroglobulin by cathepsin E

    M Shibata, H Sakai, E Sakai, K Okamoto, K Nishishita, Y Yasuda, Y Kato, K Yamamoto

    EUROPEAN JOURNAL OF BIOCHEMISTRY   270 ( 6 )   1189 - 1198   2003.3

     More details

    Language:English   Publisher:BLACKWELL PUBLISHING LTD  

    alpha(2) -Macroglobulin (alpha2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha2M and biological targets, little is known about the nature of alpha2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha2M in the endolysosome system, we examined the capacity of alpha2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha2M in the endolysosome system.

    DOI: 10.1046/j.1432-1033.2003.03479.x

    Web of Science

    PubMed

    CiNii Article

    researchmap

  • Porphyromonas gingivalisの産生するリジン特異的システインプロテアーゼ発現系の確立

    岡元邦彰, 胡錦へい, 柴田光枝, 坂井詠子, 門脇知子, 庄子幹郎, 山本健二, 中山浩次, 加藤有三

    歯科基礎医学会雑誌   45 ( 5 )   2003

  • Cathepsin E and atopic dermatitis.

    T Tsukuba, K Okamoto, K Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91   13P - 13P   2003

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • The role of propeptide of cathepsin E in its biosynthetic pathway

    Y Yasuda, K Kohmura, K Okamoto, T Tsukuba, K Yamamoto

    JAPANESE JOURNAL OF PHARMACOLOGY   88   183P - 183P   2002

     More details

    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

    researchmap

  • New functional aspects of cathepsin D and cathepsin E

    T Tsukuba, K Okamoto, Y Yasuda, W Morikawa, H Nakanishi, K Yamamoto

    MOLECULES AND CELLS   10 ( 6 )   601 - 611   2000.12

     More details

    Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:SPRINGER-VERLAG SINGAPORE PTE LTD  

    Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recently, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.

    Web of Science

    researchmap

  • Porphyromonas gingivalis proteinases as virulence determinants in progression of periodontal diseases

    Tomoko Kadowaki, Koji Nakayama, Kuniaki Okamoto, Naoko Abe, Atsuyo Baba, Yixin Shi, Dinath B. Ratnayake, Kenji Yamamoto

    Journal of Biochemistry   128 ( 2 )   153 - 159   2000.8

     More details

    Language:English   Publishing type:Book review, literature introduction, etc.  

    Porphyromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-associated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lysgingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingivalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the virulence of P. gingivalis.

    DOI: 10.1093/oxfordjournals.jbchem.a022735

    Scopus

    PubMed

    researchmap

  • 歯周病原性細菌のヘムおよびアミノ酸獲得機構

    山本健二, 岡元邦彰, 門脇知子, 安部直子, 馬場貴代, 中山浩次

    生化学   72 ( 8 )   2000

  • 歯周病原性細菌の産生するリジン特異的システインプロテアーゼの分布と機能解析

    安部直子, 門脇知子, 岡元邦彰, 岡崎真治, 浅尾哲次, 中山浩次, 山本健二

    歯科基礎医学会雑誌   42 ( 5 )   2000

  • Arginine- and lysine-specific gingipains from Porphyromonas gingivalis as major virulence factors of progressive periodontal disease.

    山本健二, 門脇知子, 岡元邦彰, 安部直子, 中山浩次

    炎症   18 ( 4 )   259 - 264   1998

  • Structural and functional characterization of periodontal pathogenic cysteine proteinases from Porphyromonas gingivalis

    K. Yamamoto, T. Kadowaki, K. Okamoto, K. Nakayama

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   42 ( 14 Suppl )   2425 - 2432   1997.10

     More details

    Publishing type:Book review, literature introduction, etc.  

    Scopus

    PubMed

    researchmap

  • Processing of exine protein and secretion protein by Porphyromonas gingivalis arginine peculiar cysteine proteinase (Arg-gingipain).

    門脇知子, 中山浩次, 吉村文信, 岡元邦彰, 山本健二

    日本分子生物学会年会プログラム・講演要旨集   19th   1996

  • Periodontal disease and pathogenic protease.

    山本健二, 門脇知子, 岡元邦彰

    モダンフィジシャン   16 ( 12 )   1996

  • Structural comparison of the pathogenicity protease gene of Porphyromonas gingivalis.

    岡元邦彰, 門脇知子, 中山浩次, 山本健二

    日本分子生物学会年会プログラム・講演要旨集   19th   1996

▼display all

Presentations

  • 新規腫瘍オルガノイド形成多元評価システムの薬剤スクリーニングへの応用

    十川千春, 江口傑徳, 石毛真行, 河合穂高, 奥舎有加, 中野敬介, 十川紀夫, 小崎健一, 岡元邦彰

    2019.12.15 

     More details

    Event date: 2019.12.15

    Presentation type:Oral presentation (general)  

    researchmap

  • Keap1遺伝子欠損はNrf2の活性化を介して破骨細胞分化を抑制する

    坂井詠子, 福間裕, 西下一久, 岡元邦彰, 筑波隆幸

    第40回日本分子生物学会年会/第90回日本生化学会大会  2017.12 

     More details

    Event date: 2017.12.6 - 2017.12.9

    researchmap

  • 口腔扁平上皮癌由来エクソソームニ含まれるプロテオームの特性

    小野喜章, 江口傑徳, 十川千春, 村上純, 藤原敏史, 笠井智成, 妹尾昌治, 佐々木朗, 小崎健一, 岡元邦彰

    第40回日本分子生物学会年会/第90回日本生化学会大会  2017.12 

     More details

    Event date: 2017.12.6 - 2017.12.9

    researchmap

  • Transcription factor Sp1 regulates the expression of the murine cathepsin E gene in gastric adenosarcoma cells

    2010 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Transcription factor Sp1 regulates the expression of the murine cathepsin E gene in gastric adenosarcoma cells

    第83回日本薬理学会年会  2010 

     More details

    Presentation type:Poster presentation  

    researchmap

  • 破骨細胞分化における鉄代謝とミトコンドリアストレスの重要性

    第33回日本鉄バイオサイエンス学会学術集会  2009 

     More details

  • カテプシンE欠損によるオートファジー低下とそれに伴うミトコンドリア機能低下と酸化ストレスの増大

    第14回日本病態プロテアーゼ学会  2009 

     More details

    Presentation type:Poster presentation  

    researchmap

  • IMPAIRMENT OF AUTOPHAGY ACCOMPANIED BY INCREASED ABERRANT MITOCHONDRIA AND OXIDATIVE STRESS IN CATHEPSIN E DEFICIENT MACROPHAGES

    2009 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損マクロファージにおけるオートファジーの低下とそれに伴うミトコンドリア機能異常と酸化ストレスの上昇

    第51回歯科基礎医学会学術大会  2009 

     More details

    Presentation type:Poster presentation  

    researchmap

  • IMPAIRMENT OF AUTOPHAGY ACCOMPANIED BY INCREASED ABERRANT MITOCHONDRIA AND OXIDATIVE STRESS IN CATHEPSIN E DEFICIENT MACROPHAGES

    5th International Symposium on Autophagy  2009 

     More details

    Presentation type:Poster presentation  

    researchmap

  • 鉄代謝を介した新規の破骨細胞分化機構

    第51回歯科基礎医学会学術大会  2009 

     More details

    Presentation type:Poster presentation  

    researchmap

  • RANKL誘導性破骨細胞分化における鉄結合蛋白の関与

    第50回歯科基礎医学会学術大会  2008 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損はマクロファージの膜蛋白質輸送異常とミトコンドリア機能異常を起こす

    第31回日本分子生物学会/第81回日本生化学会合同大会  2008 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損による遊走能および細胞接着能の低下

    第50回歯科基礎医学会学術大会  2008 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE遺伝子発現に関する転写因子Sp1

    第31回日本分子生物学会/第81回日本生化学会合同大会  2008 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損マウスにおける高脂血症および脂肪肝の誘導

    第50回歯科基礎医学会学術大会  2008 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE遺伝子発現に関与する転写因子Sp1

    第50回歯科基礎医学会学術大会  2008 

     More details

    Presentation type:Poster presentation  

    researchmap

  • CONTROL OF THE ENDOSOME/LYSOSOME SYSTEM OF MACROPHAGES BY CATHEPSIN E

    5TH General Meeting of the International Proteolysis Society  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • 赤血球におけるカテプシンEの役割

    第49回歯科基礎医学会学術大会  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • P. gingivalis感染とインスリン抵抗性に関する実験的解析

    第49回歯科基礎医学会学術大会  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • ASSOCIATION OF CATHEPSIN E WITH HOST DEFFENCE AGAINST TUMOR CELLS

    5TH General Meeting of the International Proteolysis Society  2007 

     More details

  • 成熟破骨細胞の延命と脂質ラフト形成におけるリセドロネートおよびD-PDMPの作用

    第49回歯科基礎医学会学術大会  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Cathepsin E mediates membrane trafficking in macrophages

    第80回日本薬理学会  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損によるミトコンドリア機能異常と酸化ストレス亢進

    第49回歯科基礎医学会学術大会  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • ベルベリンによる破骨細胞生存の抑制機構の解明

    第49回歯科基礎医学会学術大会  2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Cathepsin E mediates membrane trafficking in macrophages

    2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • CONTROL OF THE ENDOSOME/LYSOSOME SYSTEM OF MACROPHAGES BY CATHEPSIN E

    2007 

     More details

    Presentation type:Poster presentation  

    researchmap

  • ASSOCIATION OF CATHEPSIN E WITH HOST DEFFENCE AGAINST TUMOR CELLS

    2007 

     More details

  • Suppression of carcinogenesis and tumor growth by cathepsin E

    20th IUBMB International Congress of Biochemistry and Molecular Biology  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Porphyromonas gingivalisの血球凝集および接着性におけるHGP44の役割とC末端ペプチドの阻害効果

    第48回歯科基礎医学会学術大会  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • P. gingivalis感染の生活習慣病増悪機序について

    第48回歯科基礎医学会学術大会  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • マウスカテプシンE遺伝子発現におけるSp1の関与

    日本分子生物学会2006フォーラム  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損マクロファージにおける膜タンパク質の輸送異常

    第48回歯科基礎医学会学術大会  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Berberine inhibits RANKL, TNFa, LPS and PGN-induced mature osteoclasts survival

    第79回日本薬理学会総会  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Involvement of glycosphingolipids in LPS mediated survival of mature osteoclasts

    第79回日本薬理学会総会  2006 

     More details

  • A possible mechanism for cathepsin E-mediated tumor supression

    Satellite Meeting for the 20th IUBMB International Congress and 11th FAOBMB Congress  2006 

     More details

    Presentation type:Poster presentation  

    researchmap

  • ベルベリンによる破骨細胞形成抑制の分子機構

    第58回日本薬理学会西南部会  2005 

     More details

  • スフィンゴ糖脂質による破骨細胞内LPSシグナル伝達の制御

    第47回歯科基礎医学会学術大会  2005 

     More details

    Presentation type:Poster presentation  

    researchmap

  • ベルベリンの破骨細胞形成抑制作用のメカニズム検討

    第47回歯科基礎医学会学術大会  2005 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Aberrant antigen processing in cathepsin E-deficient dendritic cells

    第78回日本生化学会  2005 

     More details

    Presentation type:Poster presentation  

    researchmap

  • 破骨細胞におけるスフィンゴ糖脂質の役割

    第47回歯科基礎医学会学術大会  2005 

     More details

  • Cathepsin E is important for endo/lysosomal systems in macrophages against a bacterial infection.

    第78回日本薬理学会総会  2005 

     More details

  • Alterration of substance P levels in various regions of mouse brain caused by cathepsin E-deficiency

    日本薬理学会総会  2005 

     More details

  • Porphyromonas gingivalisの産生するシステインプロテアーゼ(Kgp)の活性中心の決定

    第47回歯科基礎医学会学術大会  2005 

     More details

    Presentation type:Poster presentation  

    researchmap

  • Glycosphingolipids regulate osteoclastgenesis mediated by lipopolysaccharide

    第78回日本薬理学会総会  2005 

     More details

  • Berberine Inhibits RANKL, TNF-a, LPS and Peptidoglycan (PGN)-induced Fusion of Osteoclasts

    日中医学会  2005 

     More details

    Presentation type:Poster presentation  

    researchmap

  • カテプシンE欠損が及ぼすマクロファージの機能異常

    第58回日本薬理学会西南部会  2005 

     More details

▼display all

Works

  • 破骨細胞におけるカテプシンEの役割と創薬にむけた基質分子の探究

    2009
    -
    2011

     More details

  • 骨疾患における創薬のターゲットとなりうるプロテアーゼの作用機序の解明

    2007
    -
    2009

     More details

  • 歯周病原性細菌の産生するプロテアーゼの膜輸送システムの解明

    2005

     More details

Awards

  • 日本生化学会JB論文賞

    2004  

     More details

    Country:Japan

    researchmap

Research Projects

  • フラボノイドをベースにした抗がん作用をもつサプリメントの開発

    2016.04 - 2019.03

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • ステロイドによって誘発される膜タンパク質の細胞生物学的解析

    2012.04 - 2015.03

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • 破骨細胞に関するカテプシンEの役割と創薬にむけた探求

    2011.04 - 2012.03

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • 骨疾患における創薬のターゲットとなりうるプロテアーゼの作用機序の解明

    2009.04 - 2011.03

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • 歯周病原性細菌の産生するプロテアーゼの膜輸送システムの解明

    2007.04 - 2009.03

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • 歯周病原性細菌におけるタンパク質発現機構の確立

    2005.04 - 2007.03

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • 歯周病に関わる因子における細胞生物学的研究

    2004.04 - 2005.03

    長崎大学  教育改善推進費 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • 歯周病原性細菌の産生するプロテアーゼの高次構造と輸送機構に関する研究

    2003.04 - 2005.03

    日本学術振興会科学研究費補助金  基盤研究奨励研究A 

    岡元 邦彰

      More details

    Authorship:Principal investigator  Grant type:Competitive

    researchmap

  • The role of proteinase in osteoclast formation and activation

    2001

    Grant-in-Aid for Scientific Research 

      More details

    Grant type:Competitive

    researchmap

  • 破骨細胞形成・活性化におけるプロテアーゼの役割

    2001

    科学研究費補助金 

      More details

    Grant type:Competitive

    researchmap

  • Study on transport of aspartic proteinase

    1993

      More details

    Grant type:Competitive

    researchmap

  • 歯周病原性細菌の産生するプロテアーゼに関する研究

    1993

      More details

    Grant type:Competitive

    researchmap

  • アスパラギン酸プロテアーゼの輸送機構に関する研究

    1993

      More details

    Grant type:Competitive

    researchmap

  • Study on proteinase from periodontal pathogen

    1993

      More details

    Grant type:Competitive

    researchmap

▼display all

 

Class subject in charge

  • How to start volunteer (2021academic year) Fourth semester  - 金4,金5,金6,金7

  • Regulatory science (2021academic year) Fourth semester  - 月1~3,金1~3

  • Medical law ethics and social welfare (2021academic year) special  - その他

  • Oral functions in health (2021academic year) Second semester  - 木3~4

  • Research Presentation in Oral Functional Reconstruction (2021academic year) special  - その他

  • Exercise in Dental Education (2021academic year) special  - その他

  • Exercise for dental health care (2021academic year) 1st and 2nd semester  - 火1~3

  • Dental pharmacology (2021academic year) Second semester  - 金4,金5

  • Research Projects and Practicals: Dental Pharmacology I (2021academic year) special  - その他

  • Lecture and Research Projects: Dental Pharmacology I (2021academic year) special  - その他

  • Research Projects and Practicals: Dental Pharmacology II (2021academic year) special  - その他

  • Lecture and Research Projects: Dental Pharmacology II (2021academic year) special  - その他

  • Pharmacology (2021academic year) Concentration  - その他

  • Pharmacology 1 (2021academic year) 1st semester  - 水1,水2,水3

  • Pharmacology 2 (2021academic year) Fourth semester  - 水1,水2,水3

  • Practice of pharmacology (2021academic year) Second semester  - 木4,木5,木6,木7

  • Seminar in pharmacology (2021academic year) Second semester  - 金6,金7

  • General pharmacology (2021academic year) 1st semester  - 水1,水2,水3

  • How to start volunteer (2020academic year) Fourth semester  - 金4,金5,金6,金7

  • Medical law ethics and social welfare (2020academic year) special  - その他

  • Oral functions in health (2020academic year) Second semester  - 木3,木4

  • Research Presentation in Oral Functional Reconstruction (2020academic year) Year-round  - その他

  • Oral Functional Reconstruction (2020academic year) Year-round  - その他

  • Exercise for dental health care (2020academic year) 1st and 2nd semester  - 火1,火2,火3

  • Dental pharmacology (2020academic year) Second semester  - 金4,金5

  • Research Projects and Practicals: Dental Pharmacology I (2020academic year) special  - その他

  • Lecture and Research Projects: Dental Pharmacology I (2020academic year) special  - その他

  • Research Projects and Practicals: Dental Pharmacology II (2020academic year) special  - その他

  • Lecture and Research Projects: Dental Pharmacology II (2020academic year) special  - その他

  • Pharmacology 1 (2020academic year) 1st semester  - 水1,水2,水3

  • Pharmacology 2 (2020academic year) Fourth semester  - 水1,水2,水3

  • Practice of pharmacology (2020academic year) Second semester  - 木4,木5,木6,木7

  • Seminar in pharmacology (2020academic year) Second semester  - 金6,金7

  • General pharmacology (2020academic year) 1st semester  - 水1,水2,水3

  • New development of gene engineering (2020academic year) Third semester  - 木3,木4

▼display all

 

Social Activities

  • 骨粗鬆症とその治療薬

    Role(s):Lecturer

    岡山大学  2018.8.30

     More details

    Type:Lecture

    researchmap

  • 岡山大学歯学部紹介, 歯科薬理学講義, 歯科薬理学教室での研究内容

    Role(s):Lecturer

    津山高等学校  2018.6.29

     More details

    Type:Visiting lecture

    researchmap

  • 生理学講義

    Role(s):Lecturer

    長崎柔鍼スポーツ専門学校  2008.4 - 2016.2

     More details

    Type:Other

    researchmap

  • 薬理学講義

    Role(s):Lecturer

    長崎市歯科衛生士専門学校  2003.4 - 2005.3

     More details

    Type:Other

    researchmap