Faculty Profiles - OKAMURA Hirohiko

Papers - OKAMURA Hirohiko

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Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury. Reviewed

Yishida K, Yoshida K, Fujiwara N, Seyama M, Ono K, Kawai H, Guo J, Wang Z, Weng Y, Yu Y, Uchida-Fukuhara Y, Ikegame M, Sasaki A, Nagatsuka H, Kamioka H, Okamura H, Ozaki K.

Biochimica Biophysica Acta-Molecular Basis of Disease 1867 ( 11 ) 166236 2021.11

Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis

Yilin Zheng, Ziyi Wang, Yao Weng, Heriati Sitosari, Yuhan He, Xiu Zhang, Noriko Shiotsu, Yoko Fukuhara, Mika Ikegame, Hirohiko Okamura

Scientific Reports 15 ( 1 ) 2025.3

Angiogenin-induced Osteoclastogenesis Mediates Bone Destruction in Oral Squamous Carcinoma

KASUMI AOKI, NANA YOSHITANI, NAITO KURIO, NORIE YOSHIOKA, JUMPEI TERAMACHI, MIKA IKEGAME, HIROHIKO OKAMURA, SOICHIRO IBARAGI

Anticancer Research 45 ( 3 ) 1025 - 1033 2025.3

PTPA localized in the Golgi apparatus plays an important role in osteoblast differentiation

Xinyu Zheng, Yao Weng, Ayano Satoh, Airi Tanai, Mika Ikegame, Koji Kimura, Nana Yoshitani, Xiaohua Xie, Hirohiko Okamura

Biochemical and Biophysical Research Communications 748 151329 - 151329 2025.2

High Glucose Inhibits O‐GlcNAc Transferase Translocation in Early Osteoblast Differentiation by Altering Protein Phosphatase 2A Activity

Heriati Sitosari, Yoko Fukuhara, Yao Weng, Yilin Zheng, Yuhan He, Xinyu Zheng, Mika Ikegame, Hirohiko Okamura

Journal of Cellular Physiology 240 ( 1 ) 2025.1

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

ABSTRACT

Our previous study revealed a link between O‐GlcNAc transferase (OGT) localization and protein phosphatase 2A (PP2A) activity in osteoblast. Given the association of PP2A downregulation with osteoblast differentiation, we hypothesized that OGT localization changes during this process. We examined OGT localization in MC3T3‐E1 cells undergoing differentiation under normal and high glucose conditions. Changes in PP2A activity were followed by alterations in OGT localization. Organ culture of calvaria revealed similar OGT localization changes in bone‐surrounding osteoblasts near the suture area. Furthermore, the levels of O‐GlcNAc modification in various proteins including Runt‐related transcription factor 2, Osterix, and ATP synthase subunit alpha (ATP5A) were shifted in parallel with OGT translocation. These findings suggest a regulatory role of OGT, under the influence of PP2A, during osteoblast differentiation.

DOI： 10.1002/jcp.31524

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P. gingivalis-Infected Macrophage Extracellular Vesicles Cause Adverse Pregnancy Outcomes. International journal

A Tanai, Y Fukuhara, T Eguchi, H Kawai, K Ueda, K Ochiai, M Ikegame, K Okamoto, H Okamura

Journal of dental research 104 ( 1 ) 54 - 63 2025.1

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

Periodontitis is a chronic inflammatory disease triggered by oral bacterial infection, with the bacterium Porphyromonas gingivalis being a major causative agent. The association between periodontitis and various systemic diseases has been demonstrated. Recent research has also highlighted the relationship between the aggravation of maternal periodontitis and adverse pregnancy outcomes such as preterm birth and low birth weight. However, the molecular mechanisms underlying how factors from periodontitis influence pregnancy and fetal development remain unclear. Extracellular vesicles (EVs) are nano-sized spherical particles secreted into the tissue microenvironment by various types of cells. EVs have garnered interest in recent years due to their role in intercellular communication. In the present study, we investigated whether EVs derived from P. gingivalis-infected macrophages (Pg-inf EVs) reach the feto-placental unit and influence fetal development. Through a series of in vivo experiments in mice, we demonstrated that Pg-inf EVs translocated to the feto-placental unit and impaired fetal development in size and weight. Histological analysis revealed disoriented blood vessel alignment and impaired angiogenesis in the placentas of Pg-inf EV-injected groups, indicative of compromised placental function. Proteome analysis revealed a significant decrease in Vegfr1 expression in the placentas of the experimental group. Moreover, Pg-inf EVs reduced VEGFR1 expression in cultured human vascular endothelial cells, highlighting a potential molecular mechanism through which these EVs exert their effects on placental angiogenesis. This is the first study to reveal a novel pathway in which oral bacteria-infected macrophage EVs in maternal periodontitis affect pregnancy via the feto-placental unit.

DOI： 10.1177/00220345241285132

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O‐<scp>GlcNAcylation</scp> regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum Reviewed

Yao Weng, Ziyi Wang, Heriati Sitosari, Mitsuaki Ono, Hirohiko Okamura, Toshitaka Oohashi

BioFactors 2024.10

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

Abstract

To explore the potential mechanisms which O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates osteogenesis, a publicly RNA‐seq dataset was re‐analyzed with literature‐mining and showed the primary targets of O‐GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O‐GlcNAcylation‐regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt‐KO) in MC3T3‐E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria–endoplasmic reticulum (Mito–ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt‐KO cells. Through artificial intelligence (AI)‐predicted cellular structures, the time‐lapse live cells imaging with reactive‐oxygen‐species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt‐KO cells were correlated with the Mito–ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O‐GlcNAcylation‐regulated Mito–ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O‐GlcNAcylation‐regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role.

DOI： 10.1002/biof.2131

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β-catenin binds to GSK-3β in liquid-liquid phase separation compartment in HEK293 cells Reviewed

Kato M., Tanai A., Fukuhara Y., Zheng Y., Sitosari H., Yamamoto T., Ikegame M., Okamura H.

Journal of Hard Tissue Biology 2024.10

P. gingivalis-infected macrophage EVs cause adverse pregnancy outcomes Reviewed

Tanai A., Fukuhara T., Eguchi T., Kawai K., Ueda K., Ochiai M., Ikegame M., Okamoto K., Okamura H.

Journal of Dental Research 2024.9

Mechanical stretching determines the orientation of osteoblast migration and cell division. Reviewed

Fumiko Takemoto, Yoko Uchida-Fukuhara, Hiroshi Kamioka, Hirohiko Okamura, Mika Ikegame

Anatomical science international 98 ( 4 ) 521 - 528 2023.9

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

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.

DOI： 10.1007/s12565-023-00716-8

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Neuropilin 1 (NRP1) Positively Regulates Adipogenic Differentiation in C3H10T1/2 Cells Reviewed International journal

Yaqiong Yu, Yoko Uchida-Fukuhara, Yao Weng, Yuhan He, Mika Ikegame, Ziyi Wang, Kaya Yoshida, Hirohiko Okamura, Lihong Qiu

International Journal of Molecular Sciences 24 ( 8 ) 7394 - 7394 2023.4

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

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.

DOI： 10.3390/ijms24087394

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Inhibition of protein phosphatase 2A by okadaic acid induces translocation of nucleocytoplasmic O-GlcNAc transferase Reviewed International journal

Heriati Sitosari, Ikkei Morimoto, Yao Weng, Yilin Zheng, Yoko Fukuhara, Mika Ikegame, Hirohiko Okamura

Biochemical and Biophysical Research Communications 646 50 - 55 2023.2

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

Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.

DOI： 10.1016/j.bbrc.2023.01.033

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Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling. Reviewed International journal

Haoze Yuan, Mika Ikegame, Yoko Fukuhara, Fumiko Takemoto, Yaqiong Yu, Jumpei Teramachi, Yao Weng, Jiajie Guo, Daisuke Yamada, Takeshi Takarada, Ying Li, Hirohiko Okamura, Bin Zhang

Calcified tissue international 111 ( 3 ) 331 - 344 2022.6

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

Our previous gene profiling analysis showed that the transcription cofactor vestigial-like 3 (VGLL3) gene expression was upregulated by mechanical tension in the mouse cranial suture, coinciding with accelerated osteoblast differentiation. Therefore, we hypothesized that VGLL3 plays a significant role in osteogenic differentiation. To clarify the function of VGLL3 in osteoblasts, we examined its expression characteristics in mouse bone tissue and the osteoblastic cell line MC3T3-E1. We further examined the effects of Vgll3 knockdown on osteoblast differentiation and bone morphogenetic protein (BMP) signaling. In the mouse cranial suture, where membranous ossification occurs, VGLL3 was immunohistochemically detected mostly in the nucleus of osteoblasts, preosteoblasts, and fibroblastic cells. VGLL3 expression in MC3T3-E1 cells was transient and peaked at a relatively early stage of differentiation. RNA sequencing revealed that downregulated genes in Vgll3-knockdown cells were enriched in gene ontology terms associated with osteoblast differentiation. Interestingly, most of the upregulated genes were related to cell division. Targeted Vgll3 knockdown markedly suppressed the expression of major osteogenic transcription factors (Runx2, Sp7/osterix, and Dlx5) and osteoblast differentiation. It also attenuated BMP signaling; moreover, exogenous BMP2 partially restore osteogenic transcription factors' expression in Vgll3-knockdown cells. Furthermore, overexpression of Vgll3 increased the expression of osteogenic transcription factors. These results suggest that VGLL3 plays a critical role in promoting osteoblast differentiation and that part of the process is mediated by BMP signaling. Further elucidation of VGLL3 function will increase our understanding of osteogenesis and skeletal disease etiology.

DOI： 10.1007/s00223-022-00997-7

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Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction. Reviewed International journal

Yoko Uchida-Fukuhara, Takako Hattori, Shanqi Fu, Sei Kondo, Miho Kuwahara, Daiki Fukuhara, Md Monirul Islam, Kota Kataoka, Daisuke Ekuni, Satoshi Kubota, Manabu Morita, Mika Iikegame, Hirohiko Okamura

Microorganisms 10 ( 5 ) 2022.5

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

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.

DOI： 10.3390/microorganisms10051000

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Osteoblast Jmjd3 regulates osteoclastogenesis via EphB4 and RANKL signalling Reviewed

Wang R, Luo H, Yang D, Yu B, Guo J, Shao L, Okamura H, Qiu L

2022.2

Histone Demethylase Jmjd3 Regulates the Osteogenic Differentiation and Cytokine Expressions of Periodontal Ligament Cells Reviewed

Yu B, Wang R, Luo H, Yang D, Wang S, Yu Y, Okamura H, Qiu L

Acta medica Okayama 76 ( 3 ) 281 - 290 2022.1

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

Periodontal ligament (PDL) cells are critical for the bone remodeling process in periapical lesions since they can differentiate into osteoblasts and secrete osteoclastogenesis-promoting cytokines. Post-translational histone modifications including alterations of the methylation status of H3K27 are involved in cell differentiation and inflammatory reaction. The histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes methylation of H3K27. We investigated whether Jmjd3 is involved in the osteogenic differentiation and secretion of PDL cells' inflammatory factors. Jmjd3 expression in periapical lesions was examined by immunostaining. Using siRNA specific for Jmjd3 or the specific Jmjd3 inhibitor GSK-J4, we determined Jmjd3's roles in osteogenic differentiation and cytokine production by real-time RT-PCR. The locations of Jmjd3 and NF-κB were analyzed by immunocytochemistry. Compared to healthy PDLs, the periapical lesion samples showed higher Jmjd3 expression. Treatment with GSK-J4 or Jmjd3 siRNA suppressed PDL cells' osteogenic differentiation by suppressing the expressions of bone-related genes (Runx2, Osterix, and osteocalcin) and mineralization. Jmjd3 knockdown decreased the expressions of cytokines (TNF-α, IL-1β, and IL-6) induced by lipopolysaccharide extracted from Porphyromonas endodontalis (Pe-LPS). Pe-LPS induced the nuclear translocations of Jmjd3 and NF-κB; the latter was inhibited by GSK-J4 treatment. Jmjd3 appears to regulate PDL cells' osteogenic differentiation and proinflammatory cytokine expressions.

DOI： 10.18926/AMO/63722

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Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury. Reviewed International journal

Kayo Yoshida, Kaya Yoshida, Natsumi Fujiwara, Mariko Seyama, Kisho Ono, Hotaka Kawai, Jiajie Guo, Ziyi Wang, Yao Weng, Yaqiong Yu, Yoko Uchida-Fukuhara, Mika Ikegame, Akira Sasaki, Hitoshi Nagatsuka, Hiroshi Kamioka, Hirohiko Okamura, Kazumi Ozaki

Biochimica et biophysica acta. Molecular basis of disease 1867 ( 11 ) 166236 - 166236 2021.11

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

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.

DOI： 10.1016/j.bbadis.2021.166236

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Outer membrane vesicles of Porphyromonas gingivalis: novel communication tool and strategy. Invited Reviewed International coauthorship

Japanese Dental Science Review 57 138 - 146 2021.11

Outer membrane vesicles of Porphyromonas gingivalis: Novel communication tool and strategy. Invited Reviewed International journal

Hirohiko Okamura, Katsuhiko Hirota, Kaya Yoshida, Yao Weng, Yuhan He, Noriko Shiotsu, Mika Ikegame, Yoko Uchida-Fukuhara, Airi Tanai, Jiajie Guo

The Japanese dental science review 57 138 - 146 2021.11

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

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.

DOI： 10.1016/j.jdsr.2021.07.003

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Porphyromonas gingivalis impairs placental and fetal development through macrophage-derived extracellular vesicles) Reviewed

2021 205 - 205 2021.10

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Papers - OKAMURA Hirohiko