Wiley Acta Medica Okayama 0951-6433 52 3 2026 VGLL3 Regulates DAPK2-Mediated Autophagy During Osteoblast Differentiation e70104 EN Yuhan He Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ziyi Wang Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yao Weng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Heriati Sitosari Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada Yilin Zheng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yaxin Qu Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Vestigial-like family member 3 (VGLL3), a transcriptional cofactor of the TEA domain family, has been previously identified as a regulator of osteoblast differentiation. Building upon our previous findings, we investigated VGLL3 function in MC3T3-E1 osteoblasts using an integrated approach combining transcriptomic analysis and functional assays to identify its downstream effectors and explore associated autophagy mechanisms. RNA-seq analysis of Vgll3-knockdown (shVgll3) cells identified death-associated protein kinase 2 (DAPK2), a regulator of autophagy, as a downstream effector. Autophagic activity was examined using transmission electron microscopy and western blot analysis of LC3-II and p62 proteins. The effects of Dapk2 knockdown (shDapk2) on osteoblast differentiation were evaluated using qPCR, western blotting, alkaline phosphatase staining, and Alizarin Red staining. Rapamycin treatment was used to determine whether pharmacologic activation of autophagy could restore osteoblast function. Vgll3 knockdown significantly suppressed autophagic flux, as evidenced by fewer autophagic vacuoles, decreased LC3-II accumulation, and increased p62 expression. A comparable reduction in autophagic activity was observed in shDapk2 cells and was accompanied by impaired osteoblast differentiation. Rapamycin treatment partially restored autophagy and osteogenic differentiation in Vgll3-deficient cells. Finally, overexpression of DAPK2 partially rescued autophagic activity and osteogenic differentiation in shVgll3 cells, supporting its role as a key downstream functional effector. FOXM1 was further implicated as a potential transcriptional regulator contributing to DAPK2 expression. Collectively, our findings suggest that VGLL3 may influence osteogenic differentiation in osteoblasts, potentially involving DAPK2-associated autophagy. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 1349-0079 68 1 2026 Dynamin 2 is involved in osteoblast migration by regulating the organization of F-actin 100720 EN Takumi Moriya Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University A. Surong Department of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nanami Tatsumi Department of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroshi Yamada Department of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumiko Takemoto Department of Orthodontics, Okayama University Hospital Hiroshi Kamioka Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Objectives: Dynamin, a GTPase that regulates membrane dynamics, has recently been implicated in actin cytoskeletal remodeling. This study aimed to elucidate the role of dynamin in osteoblast migration by examining the effects of dynamin inhibition on the localization and organization of F-actin and dynamin 2 in MC3T3-E1 cells. Methods: MC3T3-E1 cells were treated with dynamin inhibitors (Dyngo 4a and Dynole 34-2), and cell migration was assessed using a wound-healing assay. Fluorescent staining was performed to analyze the intracellular localization of F-actin and dynamin 2. Results: Dynamin inhibition significantly reduced the migration of MC3T3-E1 cells. Fluorescence analysis revealed a marked decrease in the accumulation and colocalization of F-actin and dynamin 2 at the protrusion edge. Additionally, dynamin inhibition suppressed the formation of lamellipodia and stress fibers while promoting the appearance of abnormal F-actin clusters in the cytoplasm. Conclusions: These findings suggest that dynamin plays an essential role in osteoblast migration by regulating actin cytoskeletal remodeling, particularly through the formation of lamellipodia and stress fibers. No potential conflict of interest relevant to this article was reported.

International Institute of Anticancer Research Acta Medica Okayama 0250-7005 45 3 2025 Angiogenin-induced Osteoclastogenesis Mediates Bone Destruction in Oral Squamous Carcinoma 1025 1033 EN KASUMI AOKI Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University NANA YOSHITANI Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University NAITO KURIO Department of Oral Surgery, Graduate School of Biomedical Sciences, Tokushima University NORIE YOSHIOKA Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University JUMPEI TERAMACHI Department of Oral Function and Anatomy, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University MIKA IKEGAME Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University HIROHIKO OKAMURA Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University SOICHIRO IBARAGI Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background/Aim: Bone destruction caused by oral cancer severely impacts patient quality of life. This study aimed to clarify the role of angiogenin (ANG) in osteoclastogenesis and oral cancer-induced bone destruction. Materials and Methods: Recombinant ANG was used to assess its effects on osteoclast formation and bone resorption activity in bone marrow cultures. ANG-knockdown oral squamous carcinoma HSC-2 cells (ANG-RNAi) were transplanted into intramedullary cavities of femurs. Bone destruction was radiologically analyzed, while angiogenesis and osteoclast induction in the surrounding area of the transplanted lesion were histologically examined. Results: Recombinant ANG promoted osteoclast formation and bone resorption activity. Transplantation of ANG-RNAi cells significantly reduced tumor growth and bone destruction properties compared to transplantation of control cells. Histological analysis revealed lower angiogenesis and fewer osteoclast induction in the ANG-RNAi cells-transplanted group. Conclusion: ANG mediates oral cancer-induced bone destruction by promoting osteoclast formation and resorption. These findings suggest that ANG could be a potential therapeutic target for suppressing tumor growth, angiogenesis, and bone destruction in oral cancer therapy. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 15 1 2025 Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis 10462 EN Yilin Zheng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Ziyi Wang Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yao Weng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Heriati Sitosari Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Yuhan He Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Xiu Zhang Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Noriko Shiotsu Comprehensive Dental Clinic, Okayama University Hospital, Okayama University Yoko Fukuhara Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hospital, Okayama University Periodontal pathogen Porphyromonas gingivalis (P. gingivalis) is believed to possess immune evasion capabilities, but it remains unclear whether this immune evasion is related to host gene alternative splicing (AS). In this study, RNA-sequencing revealed significant changes in both AS landscape and transcriptomic profile of macrophages following P. gingivalis infection with/without knockout of gingipain (a unique toxic protease of P. gingivalis). P. gingivalis infection increased the PD-L1 transcripts expression and selectively upregulated a specific coding isoform that more effectively binds to PD-1 on T cells, thereby inhibiting immune function. Biological experiments also detected AS switch of PD-L1 in P. gingivalis-infected or gingipain-treated macrophages. AlphaFold 3 predictions indicated that the protein docking compatibility between PD-1 and P. gingivalis-upregulated PD-L1 isoform was over 80% higher than another coding isoform. These findings suggest that P. gingivalis employs gingipain to modulate the AS of PD-L1, facilitating immune evasion. No potential conflict of interest relevant to this article was reported.

Society for Hard Tissue Regenerative Biology Acta Medica Okayama 1341-7649 33 4 2024 β-catenin Binds to Gsk-3β in Liquid-Liquid Phase Separation Compartment in HEK293 Cells 213 218 EN Mari Kato Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Airi Tanai Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoko Fukuhara Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Xinyu Zheng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Heriati Sitosari Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tadashi Yamamoto The Center for Graduate Medical Education (Dental Division), Okayama University Hospital Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Liquid-liquid phase separation (LLPS) has emerged as a significant mechanism for cellular organization, impacting various biological processes, including Wnt/β-catenin signaling. This study investigates the role of LLPS in the regulation of β-catenin in HEK293 cells, particularly in response to Wnt3a signaling. Our findings demonstrate that β-catenin is regulated by LLPS, forming spherical droplets indicative of this phenomenon. Fluorescence recovery after photobleaching (FRAP) assays revealed that these droplets exhibit reversible dynamics, further confirming their phase-separated nature. Importantly, treatment with Wnt3a led to an increase in β-catenin levels, while simultaneously reducing the recovery of fluorescence intensity in FRAP experiments, suggesting that enhanced Wnt signaling may stimulate the release of β-catenin from LLPS. Immunoprecipitation studies indicated that β-catenin binds to glycogen synthase kinase 3β (Gsk-3β) within the LLPS state, highlighting a potential regulatory mechanism whereby LLPS facilitates the phosphorylation and subsequent degradation of β-catenin. The addition of 1,6-hexanediol disrupted the β-catenin/Gsk-3β interaction, reinforcing the idea that LLPS plays a critical role in modulating these biochemical interactions. The findings presented in this study suggest that LLPS is not only crucial for the spatial organization of β-catenin but also serves as a regulatory mechanism for its signaling functions in the Wnt pathway. Given the association of aberrant Wnt signaling with various diseases, including cancer and neurodegenerative disorders, understanding the role of LLPS in this context may provide new insights into therapeutic strategies targeting these pathological conditions. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 24 8 2023 Neuropilin 1 (NRP1) Positively Regulates Adipogenic Differentiation in C3H10T1/2 Cells 7394 EN Yaqiong Yu Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases Yoko Uchida-Fukuhara Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yao Weng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuhan He Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ziyi Wang Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kaya Yoshida Department of Oral Healthcare Education, Institute of Biomedical Sciences, Tokushima University Graduate School Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Lihong Qiu Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1447-6959 2023 Mechanical stretching determines the orientation of osteoblast migration and cell division EN Fumiko Takemoto Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoko Uchida-Fukuhara Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroshi Kamioka Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2076-2607 10 5 2022 Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction 1000 EN Yoko Uchida-Fukuhara Department of Oral Morphology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takako Hattori Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shanqi Fu Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sei Kondo Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miho Kuwahara Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Daiki Fukuhara Department of Preventive Dentistry, Okayama University Hospital Md Monirul Islam Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kota Kataoka Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Daisuke Ekuni Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Manabu Morita Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mika Iikegame Department of Oral Morphology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta. No potential conflict of interest relevant to this article was reported.

Elsevier Ltd. Acta Medica Okayama 1882-7616 57 2021 Outer membrane vesicles of Porphyromonas gingivalis: Novel communication tool and strategy 138 146 EN Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Katsuhiko Hirota Department of Medical Hygiene, Dental Hygiene Course, Kochi Gakuen College Kaya Yoshida Department of Oral Healthcare Education, Institute of Biomedical Sciences, Tokushima University Graduate School Yao Weng Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuhan He Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Noriko Shiotsu Comprehensive Dental Clinic, Okayama University Hospital, Okayama University Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoko Uchida-Fukuhara Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Airi Tanai Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Jiajie Guo Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Extracellular vesicles (EVs) have been recognized as a universal method of cellular communications and are reportedly produced in bacteria, archaea, and eukaryotes. Bacterial EVs are often called "Outer Membrane Vesicles" (OMVs) as they were the result of a controlled blebbing of the outer membrane of gram-negative bacteria such as Porphyromonas gingivalis (P. gingivalis). Bacterial EVs are natural messengers, implicated in intra-and inter-species cell-to-cell communication among microorganism populations present in microbiota. Bacteria can incorporate their pathogens into OMVs; the content of OMVs differs, depending on the type of bacteria. The production of distinct types of OMVs can be mediated by different factors and routes. A recent study highlighted OMVs ability to carry crucial molecules implicated in immune modulation, and, nowadays, they are considered as a way to communicate and transfer messages from the bacteria to the host and vice versa. This review article focuses on the current understanding of OMVs produced from major oral bacteria, P. gingivalis: generation, characteristics, and contents as well as the involvement in signal transduction of host cells and systemic diseases. Our recent study regarding the action of P. gingivalis OMVs in the living body is also summarized. No potential conflict of interest relevant to this article was reported.

Taylor & Francis Acta Medica Okayama 2001-3078 9 1 2020 Triple knockdown of CDC37, HSP90‐alpha and HSP90‐beta diminishes extracellular vesicles‐driven malignancy events and macrophage M2 polarization in oral cancer 1769373 EN Kisho Ono Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman A. Taha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yanyin Lu Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University May Wathone Oo Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Soichiro Ibaragi Department of Oral and Maxillofacial Surgery, Okayama University Hospital Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken-Ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Akira Sasaki Department of Oral and Maxillofacial Surgery, Okayama University Hospital Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones – CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial‐mesenchymal transition (EMT), although their contribution to EVs‐mediated cell–cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer‐derived EVs (MEV) were enriched with HSP90α HSP90β and cancer‐initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV‐driven malignancy events. In metastatic oral cancer patient‐derived tumours, HSP90β was significantly accumulated in infiltrating tumour‐associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90‐enriched MEV‐induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA‐mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV‐driven malignancy events in the tumour microenvironment. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 21 24 2020 The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors 9352 EN Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yunxia Feng Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masatoshi Morimatsu Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Penggong Wei Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoko Kadowaki Department of Frontier Oral Science, Graduate School of Biomedical Sciences, Nagasaki University Eiko Sakai Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiji Naruse Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takayuki Tsukuba Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University 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. No potential conflict of interest relevant to this article was reported.

PeerJ Acta Medica Okayama 2167-8359 8 2020 Loading history changes the morphology and compressive force-induced expression of receptor activator of nuclear factor kappa B ligand/osteoprotegerin in MLO-Y4 osteocytes e10244 EN Ziyi Wang Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yao Weng Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yoshihito Ishihara Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Naoya Odagaki Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Ei Ei Hsu Hlaing Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takashi Izawa Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hiroshi Kamioka Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Background In this study, we investigated the effect of the mechanical loading history on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in MLO-Y4 osteocyte-like cells. Methods Three hours after MLO-Y4 osteocytes were seeded, a continuous compressive force (CCF) of 31 dynes/cm2 with or without additional CCF (32 dynes/cm2) was loaded onto the osteocytes. After 36 h, the additional CCF (loading history) was removed for a recovery period of 10 h. The expression of RANKL, OPG, RANKL/OPG ratio, cell numbers, viability and morphology were time-dependently examined at 0, 3, 6 and 10 h. Then, the same additional CCF was applied again for 1 h to all osteocytes with or without the gap junction inhibitor to examine the expression of RANKL, OPG, the RANKL/OPG ratio and other genes that essential to characterize the phenotype of MLO-Y4 cells. Fluorescence recovery after photobleaching technique was also applied to test the differences of gap-junctional intercellular communications (GJIC) among MLO-Y4 cells. Results The expression of RANKL and OPG by MLO-Y4 osteocytes without a loading history was dramatically decreased and increased, respectively, in response to the 1-h loading of additional weight. However, the expression of RANKL, OPG and the RANKL/OPG ratio were maintained at the same level as in the control group in the MLO-Y4 osteocytes with a loading history but without gap junction inhibitor treatment. Treatment of loading history significantly changed the capacity of GJIC and protein expression of connexin 43 (Cx43) but not the mRNA expression of Cx43. No significant difference was observed in the cell number or viability between the MLO-Y4 osteocyte-like cells with and without a loading history or among different time checkpoints during the recovery period. The cell morphology showed significant changes and was correlated with the expression of OPG, Gja1 and Dmp1 during the recovery period. Conclusion Our findings indicated that the compressive force-induced changes in the RANKL/OPG expression could be habituated within at least 11 h by 36-h CCF exposure. GJIC and cell morphology may play roles in response to loading history in MLO-Y4 osteocyte-like cells. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 4 2020 Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer 881 EN Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Manh T. Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kaya Yoshida Department of Oral Healthcare Education, Institute of Biomedical Sciences, Tokushima University Graduate School Mami Itagaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman A. Taha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Oral and Maxillofacial Surgery, Okayama University Hospital Eriko Aoyama Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-Ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University 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. No potential conflict of interest relevant to this article was reported.