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.<br> 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.<br> 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.<br> 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.<br> 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.<br> 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.<br> 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.

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.

BIOSCIENTIFICA Acta Medica Okayama 0022-0795 241 2 2019 Circadian production of melatonin in cartilage modifies rhythmic gene expression 161 173 EN Shanqi Fu Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Miho Kuwahara Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoko Uchida Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sei Kondo Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daichi Hayashi Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuji Shimomura Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Asami Takagaki Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takashi Nishida Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Maruyama Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University Mika Ikegame Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuhiko Hattori Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takako Hattori Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan, and Sox9, but inhibited Col10a1 expression in primary BALB/c mouse chondrocytes. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2, and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1, and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock. No potential conflict of interest relevant to this article was reported.

Sage Publications Acta Medica Okayama 00221554 67 2 2018 Expression of Non-collagenous Bone Matrix Proteins in Osteoblasts Stimulated by Mechanical Stretching in the Cranial Suture of Neonatal Mice 107 116 EN Mika Ikegame Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sadakazu Ejiri Department of Oral Anatomy, School of Dentistry, Asahi University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University We investigated the influence of mechanical stretching on the genetic expression pattern of non-collagenous bone matrix proteins in osteoblasts. The cranial sutures of neonatal mice were subjected to ex vivo mechanical stretching. In the non-stretched control group, as osteoblast differentiation progressed, the successive genetic expression of bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) was detected using in situ hybridization, in that order. In the stretched group, the sutures were widened, and after 24 hr of cultivation, a large number of osteoblasts and abundant new osteoid were observed on the borders of the parietal bones. All new osteoblasts expressed BSP and some of them expressed OPN, but very few of them expressed OCN. After 48 hr, more extensive presence of osteoid was noted on the borders of the parietal bones, and this osteoid was partially mineralized; all osteoblasts on the osteoid surface expressed BSP, and more osteoblasts expressed OPN than those after 24 hr cultivation. Surprisingly, many of the osteoblasts that did not express OPN, expressed OCN. This suggests that when osteoblast differentiation is stimulated by mechanical stress, the genetic expression pattern of non-collagenous proteins in the newly differentiated osteoblasts is affected. No potential conflict of interest relevant to this article was reported.