記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

添付ファイル： 65. Narahara(J Cell Physiol).pdf

DOI： 10.1002/jcp.26605

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

添付ファイル： 63. Sakamoto (Oral Dis).pdf

DOI： 10.1111/odi.12826

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

添付ファイル： 70. Komaki (Biomed Res).pdf

DOI： 10.2220/biomedres.39.169

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

添付ファイル： 67. Okusha (J Cell Biochem).pdf

DOI： 10.1002/jcb.27040

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

添付ファイル： 66. Fukuma (Clin Exp Pharmacol Physiol.pdf

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記述言語：日本語   出版者・発行元：生命科学系学会合同年次大会運営事務局  

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記述言語：日本語   出版者・発行元：生命科学系学会合同年次大会運営事務局  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

添付ファイル： 62. Iwatake (Exp Cell Res).pdf

DOI： 10.1016/j.yexcr.2017.08.025

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 60. Sakai (FASEB ).pdf

DOI： 10.1096/fj.201700177R

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

添付ファイル： 61. Yamaguchi (Cell Mol Life Sci).pdf

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 58. Inamitsu(J Appl Toxicol.pdf

DOI： 10.1002/jat.3429

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© 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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 59. Yamashita (Oral Dis).pdf

DOI： 10.1111/odi.12643

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

添付ファイル： 57. Sakai (Phytomedicine) .pdf

DOI： 10.1016/j.phymed.2016.04.002

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 56. Yoneshima (J Cellular Physiol).pdf

DOI： 10.1002/jcp.25235

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 55. Uchino (Phytother res).pdf

DOI： 10.1002/ptr.5416

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 53. Iwatake (Mol Cell Biochem).pdf

DOI： 10.1007/s11010-015-2466-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 51. Yashima (Biometals).pdf

DOI： 10.1007/s10534-015-9861-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

添付ファイル： 54. Fukuma (BioFactors).pdf

DOI： 10.1002/biof.1218

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 50. Iwatake (Phytother res).pdf

DOI： 10.1002/ptr.5333

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 51. Shimada-Sugawara (Sci Rep).pdf

DOI： 10.1038/srep09620

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 47. Kadowaki (BBRC).pdf

DOI： 10.1016/j.bbrc.2014.02.089

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 47. Kawakubo (Carcinogenesis).pdf

DOI： 10.1093/carcin/bgt373

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 48. Yamaguchi (J Appl Toxicol).pdf

DOI： 10.1002/jat.2827

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記述言語：英語   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 46. Tsukuba (Plosone).pdf

DOI： 10.1371/journal.pone.0082415

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 44. NIshishita (Gene).pdf

DOI： 10.1016/j.gene.2013.01.013

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

添付ファイル： 45. Sakai (J Pharmacol Sci).pdf

DOI： 10.1254/jphs.12243FP

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 43. Sakamoto (Toxicol In Vitro).pdf

DOI： 10.1016/j.tiv.2012.05.005

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 42. Fumimoto (J Pharmacol Sci).pdf

DOI： 10.1254/jphs.11212FP

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 41. Narahara (Cell Tissue Res).pdf

DOI： 10.1007/s00441-012-1325-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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.

添付ファイル： 40. Okamoto (j Biochem).pdf

DOI： 10.1093/jb/mvr135

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

添付ファイル： 39. Sakai (J Cell Biochem).pdf

DOI： 10.1002/jcb.23372

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：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

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DOI： 10.1515/BC.2011.060

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DOI： 10.1093/jb/mvj132

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

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DOI： 10.1074/jbc.274.25.17955

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.273.44.29072

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.273.33.21225

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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DOI： 10.1074/jbc.270.40.23619

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1006/abbi.1995.1123

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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

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記述言語：英語  

DOI： 10.1016/j.ejphar.2007.11.013

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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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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記述言語：英語  

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

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記述言語：英語  

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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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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

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記述言語：英語  

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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語  

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

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記述言語：英語  

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

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   出版者・発行元：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

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：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.

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等  

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

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DOI： 10.2492/jsir1981.18.259

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