Faculty Profiles - OKAMOTO Kuniaki

MISC - OKAMOTO Kuniaki

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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

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

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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

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

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

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

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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

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

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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

Language：English Publisher：ELSEVIER SCIENCE BV

DOI： 10.1016/j.ejphar.2007.11.013

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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

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

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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

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

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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

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

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

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

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

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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

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

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

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.

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Berberine inhibits RANKL-, TNF alpha-, LPS- and PGN-induced mature osteoclasts survival

JP Hu, K Nishishita, E Sakai, K Okamoto, Y Kato

JOURNAL OF PHARMACOLOGICAL SCIENCES 100 268P - 268P 2006

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MISC - OKAMOTO Kuniaki