Language：Japanese   Publisher：(公社)日本生化学会  

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DOI： 10.1002/cbf.3745.

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

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Language：English   Publisher：(一社)日本癌学会  

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

Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.

DOI： 10.3390/ijms23126619

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cbf.3691

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cbf.3691

Language：Japanese   Publisher：日本口腔組織培養学会  

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Clinical Investigation  

DOI： 10.1172/jci.insight.148960

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays a pivotal role in folding, activating and assembling a variety of client proteins. In addition, HSP90 has recently emerged as a crucial regulator of vesicular transport of cellular proteins. In our previous study, we revealed Rab11b negatively regulated osteoclastogenesis by promoting the lysosomal proteolysis of c-fms and RANK surface receptors via the axis of early endosome-late endosome-lysosomes. In this study, using an in vitro model of osteoclasts differentiated from murine macrophage-like RAW-D cells, we revealed that Rab11b interacted with both HSP90 isoforms, HSP90 alpha (HSP90α) and HSP90 beta (HSP90β), suggesting that Rab11b is an HSP90 client. Using at specific blocker for HSP90 ATPase activity, 17-allylamino-demethoxygeldanamycin (17-AAG), we found that the HSP90 ATPase domain is indispensable for maintaining the interaction between HSP90 and Rab11b in osteoclasts. Nonetheless, its ATPase activity is not required for regulating the turnover of endogenous Rab11b. Interestingly, blocking the interaction between HSP90 and Rab11b by either HSP90-targeting small interfering RNA (siHSP90) or 17-AAG abrogated the inhibitory effects of Rab11b on osteoclastogenesis by suppressing the Rab11b-mediated transport of c-fms and RANK surface receptors to lysosomes via the axis of early endosome-late endosome-lysosomes, alleviating the Rab11b-mediated proteolysis of these surface receptors in osteoclasts. Based on our observations, we propose a HSP90/Rab11b-mediated regulatory mechanism for osteoclastogenesis by directly modulating the c-fms and RANK surface receptors in osteoclasts, thereby contributing to the maintenance of bone homeostasis.

DOI： 10.1016/j.bbamcr.2021.119096

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500–3000 nm) reminiscent of apoptosomes, (ii) EVs (100–500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80–300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20–200 nm) containing unidentified vesicles/particles, and (v) EVs (10–70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producing a GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.

DOI： 10.3390/cells10061328

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Authorship：Last author   Publisher：MDPI AG  

Tumor-associated macrophages are a key component in the tumor microenvironment, secreting extracellular vesicles (EVs) such as exosomes and other various factors for intercellular communication. However, macrophage-derived EVs heterogeneity and their cytotoxicity to cancer cells has not been well understood. Here, we aimed to separately isolate various types of macro-phage-EVs by size exclusion chromatography (SEC) method and investigate EV transmission and cytotoxicity to oral cancer cells. For fluorescence-labeling of cellular and EV membranes, palmitoylation signal-fused GFP and tdTomato were expressed in THP-1 monocytic cells and HSC-3 oral cancer cells, respectively. We found that fluorescence-labeled EVs secreted by macrophages were highly transmissive to oral cancer cells than those from parental monocytic cells. In a co-culture system and conditioned medium (CM), a macrophage-secreted unidentified factor was cytotoxic to oral cancer cells. We fractionated macrophage-derived EVs by the SEC method and performed western blotting to characterize various EV types. Three fractions were characterized: small exosomes (EXO-S: < 50 nm) fraction containing HSP90α, HSP90β, CD63 (EV marker) and β-actin; large exosomes (EXO-L: 50-200 nm) fraction containing CD9 (EV marker) and HSP90β; large EVs (100-500 nm) fraction. Notably, the macrophage-derived small exosomes fraction was cytotoxic to oral cancer cells, while large exosomes and large EVs were not. There-fore, it was implicated that macrophage-derived small exosomes are cytotoxic with high trans-mission potential to cancer cells.

DOI： 10.20944/preprints202104.0464.v1

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/2211-5463.13133

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/2211-5463.13133

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

DOI： 10.3390/cells10020344

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Authorship：Last author   Publisher：MDPI AG  

Researchers have developed and used several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids. Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anticancer drugs have been done in 2D culture systems, where-as 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aim to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyro-sine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter tumoroid growth of metastatic colorectal cancer (mCRC). To establish a liquid-based 3D multiplexing reporter assay system, LuM1 (a murine mCRC cell line) was stably transfected with the Mmp9 promoter-driven ZsGreen reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate (NCP), a 3D culture device. The larger tumoroids were not sensitive to Cisplatin and ex-pressed ABCG2 (a marker of cancer stem cells, a.k.a. a drug efflux transporter), whereas smaller cell-aggregates were more sensitive to Cisplatin. Both Imatinib and Cisplatin significantly in-creased tumoroid growth (larger than 300 μm2) and Mmp9 promoter activity and were not cytotoxic to the mCRC tumoroids. On the other hand, 5-FU was cytotoxic to the tumoroids and significantly inhibited tumoroid growth, although not completely. Thus, platinum resistance and imatinib resistance in mCRC were modeled using the liquid-based 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

DOI： 10.20944/preprints202012.0582.v1

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

DOI： 10.3390/ijms21249352

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes–late endosomes–lysosomes in osteoclasts.

DOI： 10.3390/cells9112384

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

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Publishing type：Research paper (scientific journal)  

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 β (GSK-3β) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.

DOI： 10.1016/j.bbadis.2020.165731

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2020.05.088

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

DOI： 10.3390/cancers12051260

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Language：English  

DOI： 10.1038/s41423-020-0413-z

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Publishing type：Research paper (scientific journal)  

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.

DOI： 10.3390/cancers12040881

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Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90α was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90α, and HSP90β was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer.

DOI： 10.3390/cells9030755

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

DOI： 10.3390/cancers12020523

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Matrix metalloproteinase 3 (MMP3) plays multiple roles in pro-tumorigenic proteolysis and in intracellular transcription. These include inducing connective tissue growth factor [CTGF, also known as cellular communication network factor 2 (CCN2)] and prompting a new definition of MMP3 as a moonlighting metalloproteinase. Members of the MMP family have been found within tumor-derived extracellular vesicles (EVs) such as oncosomes or exosomes. We here investigated the roles of MMP3-rich oncosomes in tumor progression, molecular transmission, and gene regulation. MMP3 and CCN2/CTGF were significantly co-expressed in tumor samples derived from patients suffering from colorectal adenocarcinoma. We found that oncosomes derived from a rapidly metastatic colon cancer cells (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich oncosomes were highly transmissive into recipient cells and were pro-tumorigenic in an allograft mouse model. Oncosome-derived MMP3 was transmissive into recipient cell nuclei, trans-activated CCN2/CTGF promoter, and induced CCN2/CTGF production at 1 to 6 hours after the addition of oncosomes to culture media. In addition, CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects, including inhibition of migration and invasion of LuM1 cells in vitro, inhibition of tumor growth in vivo, and reduction of CCN2/CTGF and its promoter activity in vitro. These data newly demonstrate that the oncosome-derived moonlighting metalloproteinase promotes metastasis and is pro-tumorigenic at distant sites as well as a transmissive trans-activator for the cellular communication network gene.

DOI： 10.20944/preprints202002.0281.v1

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Publisher：MDPI AG  

Tumor cells exhibit a resistance-associated secretory phenotype involving extracellular vesicles (EVs) and heat shock proteins (HSPs). This response occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as in free form as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in stressome release, epithelial-to-mesenchymal transition (EMT), and tumor progression. A number of HSP family members and their common receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein species. The small EVs (30 to 200 nm in size, potentially exosomes) were released even in a non-heated condition from the prostate cancer cells, whereas EMT-coupled release of EVs (200 to 500 nm, likely ectosomes) with associated HSP90α was increased after heat shock stress (HSS). Lactate dehydrogenase, a marker of membrane leakage/damage of cells, was also released upon HSS from the prostate cancer cells. During this stress response, intracellular CDC37 was also transcriptionally inducible by heat shock factor 1, and knockdown of CDC37 decreased EMT-coupled release of EVs. Triple knockdown of CDC37, HSP90α, and HSP90β was required for efficient reduction of the chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, the data indicated that CDC37 and HSP90 are essential for stressome release and for tumorigenesis in resistant cancer.

DOI： 10.20944/preprints202002.0148.v1

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Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.1080/20013078.2020.1769373

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

Osteoporosis disturbs the balance of bone metabolism, and excessive bone resorption causes a decrease in bone density, thus increasing the risk of fracture. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin contained in green tea. EGCG has a variety of pharmacological activities. Recently, it was reported that EGCG inhibits osteoclast differentiation, but the details of the mechanism underlying the EGCG-mediated suppression of osteoclastogenesis are unknown. In this study, we investigated the effects of EGCG on several signaling pathways in osteoclastogenesis. EGCG suppressed the expression of the nuclear factor of activated T cells cytoplasmic-1 (NFATc1), the master regulator of osteoclastogenesis. EGCG decreased the expression of cathepsin K, c-Src, and ATP6V0d2 and suppressed bone resorption. We also found that EGCG upregulated heme oxygenase-1 (HO-1) and suppressed the extracellular release of high-mobility group box 1 (HMGB1). In addition, EGCG decreased the expression of the receptor for advanced glycation end products (RAGE), which is the receptor of HMGB1, in osteoclastogenesis. In summary, our study showed that EGCG could inhibit osteoclast differentiation through the downregulation of NFATc1 and the suppression of the HO-1-HMGB1-RAGE pathway. EGCG might have the potential to be a lead compound that suppresses bone resorption in the treatment of osteoporosis.

DOI： 10.2220/biomedres.41.269

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Extracellular vesicles (EVs), such as exosomes or oncosomes are released with molecules unfavorable for survival from cells. In addition, accumulating evidence has shown that tumor cells often eject anti-cancer drugs such as chemotherapeutics and targeted drugs within EVs, a novel mechanism of drug resistance. The EV-releasing, drug resistance phenotype is often coupled with cellular dedifferentiation and transformation, cells undergoing epithelial-mesenchymal transition (EMT) and taking on a cancer stem cell phenotype. Recent studies have shown that the release of EVs is also involved in immunosuppression. The concept of the resistance-associated secretory phenotype (RASP) is reviewed herein.

DOI： 10.20944/preprints201912.0386.v1

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Language：Japanese   Publisher：岡山歯学会  

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

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

CXCR4 is a chemokine receptor crucial in tumor progression, although the angiogenic role of CXCR4 in oral squamous cell carcinoma (OSCC) has not been investigated. Here we show that CXCR4 is crucial for tumor angiogenesis, thereby supporting tumor survival in OSCC. Immunohistochemistry on human clinical specimens revealed that CXCR4 and a tumor vasculature marker CD34 were co-distributed in tumor vessels in human OSCC specimens. To uncover the effects of CXCR4 inhibition, we treated the OSCC-xenografted mice with AMD3100, so-called plerixafor, an antagonist of CXCR4. Notably, we found a unique pathophysiological structure defined as tumor angiogenic inhibition triggered necrosis (TAITN), which was induced by the CXCR4 antagonism. Treatment with AMD3100 increased necrotic areas with the induction of hypoxia-inducible factor-1a in the xenografted tumors, suggesting that AMD3100-induced TAITN was involved in hypoxia and ischemia. Taken together, we demonstrated that CXCR4 plays a crucial role in tumor angiogenesis required for OSCC progression, whereas TAITN induced by CXCR4 antagonism could be an effective anti-angiogenic therapeutic strategy in OSCC treatment.

DOI： 10.3390/cells8070761

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Epidermal growth factor (EGF) is overexpressed in many cancers and is associated with worse prognosis. EGF binds to its cell surface receptor (EGFR), which induces EGFR phosphorylation. Phosphorylated EGFR (p-EGFR) is translocated into the nucleus, which increases cancer cell activity. Nicotine, which is one of the main components of tobacco, is absorbed through pulmonary alveoli and mucosal epithelia in the head and neck region by smoking and moves into the blood. Nicotine in blood binds to nicotinic acetyl-choline receptor (nAChR) in the central nervous system and serves a crucial role in tobacco addiction. Although nAChR localization is thought to be limited in the nervous system, nAChR is present in a wide variety of non-neuronal cells, including cancer cells. Recent studies suggest that nicotine contributes to the metastasis and resistance to anti-cancer drugs of various cancer cells. However, it remains unknown whether head and neck squamous cell carcinoma (HNSCC) cells can utilize nicotine-nAChR signaling to metastasize and acquire resistance to anti-cancer drugs, even though the mucosal epithelia of the head and neck region are the primary sites of exposure to tobacco smoke. To the best of our knowledge, the present study is the first to demonstrate the role of nicotine in metastasis and anti-EGFR-therapy resistance of HNSCC. The present findings demonstrated that nicotine increased proliferation, migration, invasion, p-EGFR nuclear translocation and protein kinase B (Akt) phosphorylation in HNSCC cells. It was also demonstrated that nicotine restored cetuximab-inhibited proliferation, migration and invasion of HNSCC cells. Finally, an in vivo experiment revealed that nicotine increased lymph node metastasis of xenografted tumors, whereas an nAChR inhibitor suppressed lymph node metastasis and p-EGFR nuclear localization of xenografted tumors. Taken together, these results demonstrated that nicotine induced nuclear accumulation of p-EGFR, and activation of Akt signaling. These signaling pathways elevated the activities of HNSCC cells, causing lymph node metastasis and serving a role in cetuximab resistance.

DOI： 10.3892/ijo.2018.4631

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Language：Japanese   Publisher：Japanese Pharmacological Society  

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

DOI： 10.1254/jpssuppl.92.0_2-yia-26

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Language：Japanese   Publisher：Japanese Pharmacological Society  

Cancer is one of the most serious diseases all over the world, especially metastasis and drug resistance are leading causes of death. There is an urgent need to establish new strategies for drug discovery. Success in the drug discovery depends on the development of appropriate tumor models that correspond closely to native tumor situation. Matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis and are regulators of tumor milieu. The cancer stem cell model fits well with tumorigenesis, metastasis and drug resistance. We have shown that cancer cell aggregation led to hypoxic tumoroids with marked upregulation of reprogramming and stemness genes as increased cancer stem cell using a 3D culture system. In the present study, we established a novel MMP9 promoter-driven cell-based reporter system using a rapidly metastatic colon cancer cell in the 3D culture system that evaluates cancer stemness and invasiveness. We used a concept of drug repositioning-using known molecules for new indications. We selected several compounds with inhibition to both tumoroid formation and MMP9 promoter activity. One of the compounds inhibited primary tumor formation, invasion and metastasis.

DOI： 10.1254/jpssuppl.92.0_1-p-096

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Publishing type：Part of collection (book)   Publisher：Springer International Publishing  

DOI： 10.1007/978-3-030-23158-3_1

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Language：Japanese   Publisher：岡山歯学会  

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Overexpression and increased signaling from the epidermal growth factor receptor (EGFR) often changes oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed the roles of OSCC-derived extracellular vesicles (EVs), including exosomes in the trafficking of cetuximab and in epithelial-mesenchymal transition (EMT) of epithelial cells. OSCC cells abundantly expressed EGFR, which was secreted from cells with OSCC-EVs upon EGF stimulations. The OSCC-EGFR-EVs were then able to enter into and transform epithelial cells leading to increased mesenchymal traits with increased vimentin and spindle-like shapes. EGF priming of OSCC cells further increased this EMT-initiating effect of the OSCC-EVs. The internalization and pro-EMT effects of the OSCC-EVs were largely blocked by cetuximab. Thus, OSCC-derived EVs transform normal epithelial cells into a mesenchymal phenotype and anti-EGFR therapeutic antibody cetuximab inhibits such a carcinogenic effect of the OSCC-EVs.

File： 71. Fujiwara (Oral Oncol).pdf

DOI： 10.1016/j.oraloncology.2018.09.030

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

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

DOI： 10.3389/fonc.2018.00376

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

File： 69. Fujiwara (BBRC).pdf

DOI： 10.1016/j.bbrc.2018.07.035

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

File： 68. Ono (J Cell Biochem).pdf

DOI： 10.1002/jcb.27039

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

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

DOI： 10.1111/1440-1681.12941

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

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

DOI： 10.1371/journal.pone.0191109

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

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

DOI： 10.1002/jcp.26605

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

File： 70. Komaki (Biomed Res).pdf

DOI： 10.2220/biomedres.39.169

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

File： 67. Okusha (J Cell Biochem).pdf

DOI： 10.1002/jcb.27040

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

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

File： 63. Sakamoto (Oral Dis).pdf

DOI： 10.1111/odi.12826

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

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

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

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

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

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

File： 62. Iwatake (Exp Cell Res).pdf

DOI： 10.1016/j.yexcr.2017.08.025

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

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

File： 60. Sakai (FASEB ).pdf

DOI： 10.1096/fj.201700177R

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

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

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

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

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

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

File： 58. Inamitsu(J Appl Toxicol.pdf

DOI： 10.1002/jat.3429

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Authorship：Last author   Language：English   Publisher：ELSEVIER SCIENCE BV  

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

File： 59. Yamashita (Oral Dis).pdf

DOI： 10.1111/odi.12643

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

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

File： 57. Sakai (Phytomedicine) .pdf

DOI： 10.1016/j.phymed.2016.04.002

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

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

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

DOI： 10.1002/jcp.25235

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

File： 55. Uchino (Phytother res).pdf

DOI： 10.1002/ptr.5416

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

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

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

DOI： 10.1007/s11010-015-2466-3

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

File： 51. Yashima (Biometals).pdf

DOI： 10.1007/s10534-015-9861-9

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

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

File： 54. Fukuma (BioFactors).pdf

DOI： 10.1002/biof.1218

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

File： 50. Iwatake (Phytother res).pdf

DOI： 10.1002/ptr.5333

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

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

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

DOI： 10.1038/srep09620

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

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

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

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

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

File： 47. Kadowaki (BBRC).pdf

DOI： 10.1016/j.bbrc.2014.02.089

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

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

File： 47. Kawakubo (Carcinogenesis).pdf

DOI： 10.1093/carcin/bgt373

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

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

DOI： 10.1002/jat.2827

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Language：English   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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

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

File： 46. Tsukuba (Plosone).pdf

DOI： 10.1371/journal.pone.0082415

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

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

File： 44. NIshishita (Gene).pdf

DOI： 10.1016/j.gene.2013.01.013

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

File： 45. Sakai (J Pharmacol Sci).pdf

DOI： 10.1254/jphs.12243FP

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

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

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

DOI： 10.1016/j.tiv.2012.05.005

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

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

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

DOI： 10.1254/jphs.11212FP

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

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

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

DOI： 10.1007/s00441-012-1325-8

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

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

File： 40. Okamoto (j Biochem).pdf

DOI： 10.1093/jb/mvr135

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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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File： 39. Sakai (J Cell Biochem).pdf

DOI： 10.1002/jcb.23372

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

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

DOI： 10.1016/j.job.2011.10.001

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Language：English   Publisher：Walter de Gruyter GmbH  

DOI： 10.1515/BC.2011.060

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DOI： 10.1093/jb/mvj132

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Other Link： http://search.jamas.or.jp/link/ui/2007054591

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

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

DOI： 10.1093/oxfordjournals.jbchem.a022826

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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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Language：Japanese   Publisher：(公社)日本生化学会  

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DOI： 10.1074/jbc.273.44.29072

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DOI： 10.1074/jbc.273.33.21225

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

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

DOI： 10.1093/oxfordjournals.jbchem.a021937

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Cathepsin E (CE) is a glycosylated intracellular aspartic proteinase. In this study, the role of N-glycosylation in intracellular trafficking and stability of CE was investigated in human gastric adenocarcinoma cells expressing endogenous CE (AGS-1) and recombinant normal rat kidney (NRK) cells transfected with rat CE cDNA. When these cells were treated with tunicamycin, a patent inhibitor for N-glycosylation, the initially synthesized unglycosylated proCE with an apparent molecular mass of 43 kDa was rapidly disappeared from the cells. Since secretion into the medium was not observed, the unglycosylated CE was suggested to be degraded within these cells. The amino acid sequence predicted from the cDNA revealed that rat CE (RCE) contained two potential N-glycosylation sites in the NH2-terminal (Asn(73)-Phe-Thr(75)) and COOH-terminal (Asn(305)-Val-Tnr(307)) portions. To further elucidate the significance of N-glycosylation on the stability of CE, we constructed the N-glycosylation-deficient mutants of RCE by site-directed mutagenesis (Thr(75)-->Ala(75) or Asn(305)-->Gln(305)) and expressed in NRK cells. In contrast to unglycosylated RCE generated by tunicamycin treatment, each mutant was stably retained in the cells and converted into a fully catalytically active form by a brief acid treatment in a similar manner as the wild-type CE. Pulse-chase experiments with S-35.methionine also revealed that there was no significant difference in the half-lives between each mutant and the wild-type CE in the cells. The results suggest that the deficiency of either potential N-glycosylation site alone does not affect the intracellular stability of RCE.

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Cathepsin E (CE) is an intracellular aspartic proteinase that exists mainly as a homodimer with a molecular mass of about 82 kDa and partly as a monomer of 42 kDa. In contrast to the lysosomal aspartic proteinase cathepsin D (CD), CE has a limited distribution in some animal species and is normally located in non-lysosomal compartments such as the plasma membrane, the endocytic organelles, and the endoplasmic reticulum (ER). The intracellular localization appears to be dependent on cell types expressing this enzyme. Our recent studies with pulse-chase kinetic analysis in Various cell types revealed that CE was initially synthesized as a larger precursor and underwent different proteolytic and glycolytic processing depending on cell types. This paper describes three different processing systems of CE in certain cell types. The biosynthesis and processing were studied by pulse-chase analysis with S-35-methionone and immunocytochemistry using specific antibodies to CE. In primary cultures of rat peritoneal macrophages and brain microglial cells, CE was first synthesized as a proenzyme with high-mannose-type oligosaccharide chains and subsequently processed to the mature form bearing complex-type oligosaccharides via the intermediate form within a 4 h chase period. The maturation of proCE in these cells was completely blocked by bafilomycin A1, a potent inhibitor of a vacuolar type of H+-ATPase, indicating that the maturation of CE precursor occurs in cellular acidic compartments. In rat thymocytes and mouse Friend erythroleukemia cells, the initially synthesized proCE was processed to the intermediate form within a 6 h chase period, but scarcely processed to the mature form even after a 48 h chase. However, the CE precursors in these cells became resistant to digestion by endo-beta-N-acetylglucosaminidase (Endo H) within several hours of synthesis. Interestingly, CE in thymocytes from dexamethasone-treated rats was found to be greatly processed to the mature form. In recombinant CHO and NRK cells expressing exogenous CE, the initially synthesized proenzyme was processed to the intermediate form, but not to the mature form, which did not undergo glycolytic processing.

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

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

DOI： 10.1093/oxfordjournals.jbchem.a021426

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Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase consisting of two identical subunits with a molecular mass of approximately 42 kDa and has a unique subcellular distribution in various rat tissues. In this study, we determined the complete amino acid sequence of rat spleen CE and examined the activation mechanism of the proenzyme purified from this tissue. Two cDNA clones encoding rat CE, termed pTNO5 and pTN1, were isolated. Comparison of the amino acid sequences predicted from the respective cDNA sequences revealed that they were essentially identical, except that pTN1 lacked a sequence corresponding to residues Tyr293-Pro325 of the longer cDNA clone (pTN05). Based on the structural analysis of purified enzyme forms, the CE precursor was found to comprise a signal peptide, a prosequence, and a mature protein region of 19, 39, and 337 (pTN05) or 304 (pTN1) residues, respectively. Despite a high degree of similarity in the overall structure of CE between rat and other mammalian species, the first 11 residues in the NH2-terminal sequence of rat mature enzyme were significantly different from those of other species. The purified pro-CE was analyzed for its conversion to the mature form. The results indicated that the maximal conversion occurred at pH 3.0-4.0 in a temperature- and time-dependent manner by autocatalytic cleavage at the site between Phe39-Ser40. This conversion was highly dependent on the protein concentrations of pro-CE and delayed by the presence of exogenous substrates, suggesting the predominance of intermolecular reaction for its conversion to the mature form. © 1995 Academic Press.

DOI： 10.1006/abbi.1995.1441

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DOI： 10.1074/jbc.270.40.23619

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DOI： 10.1006/abbi.1995.1123

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Language：English   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

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

DOI： 10.1111/j.1365-2591.2009.01598.x

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

DOI： 10.1016/j.joen.2008.02.018

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

DOI： 10.1016/j.ejphar.2007.11.013

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Berberine, 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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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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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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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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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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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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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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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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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

DOI： 10.1074/jbc.M413544200

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

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

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Language：English   Publisher：WALTER DE GRUYTER & CO  

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

DOI： 10.1515/BC.2004.135

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Language：Japanese   Publisher：歯科基礎医学会  

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Language：Japanese   Publisher：歯科基礎医学会  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

DOI： 10.1074/jbc.M310013200

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

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Language：English   Publisher：OXFORD UNIV PRESS  

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

DOI： 10.1093/jh/mvg216

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Language：Japanese   Publisher：歯科基礎医学会  

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Language：Japanese   Publisher：歯科基礎医学会  

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Language：Japanese   Publisher：歯科基礎医学会  

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Language：Japanese   Publisher：歯科基礎医学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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

DOI： 10.1254/jphs.91.285

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Language：English   Publisher：BLACKWELL PUBLISHING LTD  

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

DOI： 10.1046/j.1432-1033.2003.03479.x

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

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Language：Japanese   Publisher：歯科基礎医学会  

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

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Language：Japanese   Publisher：共立出版  

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Publishing type：Book review, literature introduction, etc.  

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

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

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

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

DOI： 10.1093/oxfordjournals.jbchem.a022735

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Language：Japanese   Publisher：(公社)日本生化学会  

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DOI： 10.2492/jsir1981.18.259

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Language：English   Publisher：I O S PRESS  

We have studied the role of a novel class of cysteine proteinases from the oral anaerobic bacterium Porphyromonas gingivalis in periodontal disease progression. Two unique cysteine proteinases, so termed Arg-gingipain (RGP) and Lys-gingipain (KGP) due to their peptide cleavage specificity at arginine and lysine residues, respectively, have been implicated as major virulence factors in periodontal disease. Both enzymes are shown to be involved in the direct destruction of periodontal tissues and the disruption of normal host defense mechanisms. These enzymes have also been implicated in the hemagglutinating activity of the organism. To determine the structuraI, genetic, and functional relationships between the two enzymes, we have cloned the RGP- and KGP-encoding genes and ultimately constructed the rgp- or kgp-deficient mutants from the organism. In the course of this study, RGP has been shown to be encoded by two separate genes (rgpA and rgpB) on the chromosome of the organism. Deduced amino acid sequences of both RGP- and KGP-encoding genes revealed that the precursors of both enzymes were composed of at least four domains: the signal peptide, the NH2-terminal prosequence, the proteinase domain, and the COOH-terminal hemagglutinin domain. Although both enzymes were apparently different in the amino acid sequences of the NH2-terminal prosequences and the proteinase domains, their COOH-terminal hemagglutinin domains exhibited significant similarity. Analysis of the rgpA rgpB double mutant confirmed that RGP was a major virulence factor of the organism. However, the disruption of the KGP gene did not affect either the bactericidal activity of polymorphonucler cells or the hemagglutinin activity of the organism.

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