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

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

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

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

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

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

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

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

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

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

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

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

2021 205 - 205 2021.10

O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study Reviewed

Yao Weng, Ziyi Wang, Yoko Fukuhara, Airi Tanai, Mika Ikegame, Daisuke Yamada, Takeshi Takarada, Takashi Izawa, Satoru Hayano, Kaya Yoshida, Hiroshi Kamioka, Hirohiko Okamura

BioFactors 2021.8

The role of extracellular vesicles throughout normal pregnancy and in relation to oral bacteria Reviewed

Journal of Oral Biosciences 63 ( 1 ) 14 - 22 2021.3

The role of extracellular vesicles throughout normal pregnancy and in relation to oral bacteria. Reviewed

Tanai A, Okamura H.

Journal of Oral Biosciences 63 ( 1 ) 14 - 22 2021.1

O-GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics-oriented study. Reviewed

Weng Y, Wang Z, Fukuhara Y, Tanai A, Ikegame M, Yamada D, Takarada T, Izawa T, Hayano S, Yoshida K, Kamioka H, Okamura H.

BioFactors 2021

The inhibitory role of Rab11b in osteoclastogenesis through triggering lysosome-induced degradation of c-Fms and RANK surface receptors. Reviewed

Tran MT, Okusha Y, Feng Y, Morimatsu M, Wei P, Sogawa C, Eguchi T, Kadowaki T, Sakai E, Okamura H, Naruse K, Tsukuba T, Okamoto K.

International Journal of Molecular Sciences 21 ( 24 ) 9352 2020.12

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors Reviewed

Manh Tien Tran, Yuka Okusha, Yunxia Feng, Masatoshi Morimatsu, Penggong Wei, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Hirohiko Okamura, Keiji Naruse, Takayuki Tsukuba, Kuniaki Okamoto

International Journal of Molecular Sciences 21 ( 24 ) 9352 - 9352 2020.12

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

Loading history changes the morphology and compressive force-induced expression of receptor activator of nuclear factor kappa B ligand/osteoprotegerin in MLO-Y4 osteocytes. Reviewed

Wang Z, Weng Y, Ishihara Y, Odagaki N, Ei Hsu Hlaing E, Izawa T, Okamura H, Kamioka H.

Peer J 8 e10244 2020.11

Loading history changes the morphology and compressive force-induced expression of receptor activator of nuclear factor kappa B ligand/osteoprotegerin in MLO-Y4 osteocytes Reviewed

Ziyi Wang, Yao Weng, Yoshihito Ishihara, Naoya Odagaki, Ei Ei Hsu Hlaing, Takashi Izawa, Hirohiko Okamura, Hiroshi Kamioka

PeerJ 8 e10244 - e10244 2020.11

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

<sec>
<title>Background</title>
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.

</sec>
<sec>
<title>Methods</title>
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.

</sec>
<sec>
<title>Results</title>
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.

</sec>
<sec>
<title>Conclusion</title>
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.

</sec>

DOI： 10.7717/peerj.10244

Other Link： https://peerj.com/articles/10244.xml

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium. Reviewed International coauthorship

Guo J, Wang Z, Weng Y, Yuan H, Yoshida K, Ikegame M, Uchibe K, Kamioka H, Ochiai K, Okamura H, Qiu L.

Cellular Signaling 75 1097450 2020.11

Outer membrane vesicles derived from porphylomonas gingivalis induced cell death with disruption of tight junctions in human lung epithelial cells. Reviewed International coauthorship

He Y, Shiotsu N, Uchida-Fukuhara Y, Guo J, Weng Y, Ikegame M, Wang Z, Ono K, Kamioka H, Torii Y, Sasaki A, Yoshida K, Okamura H.

Archives of Oral Biology 118 104841 2020.10

Outer membrane vesicles derived from Porphyromonas gingivalis induced cell death with disruption of tight junctions in human lung epithelial cells. Reviewed International journal

Archives of oral biology 118 104841 - 104841 2020.10

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

OBJECTIVE: Porphyromonas gingivalis (P. gingivalis) is a major bacterium responsible for the progression of periodontitis. P. gingivalis produces small vesicles called outer membrane vesicles (OMVs) containing virulence factors. Increasing evidence suggests a close relationship between periodontitis and respiratory system diseases, such as aspiration pneumonia. However, little is known about whether P. gingivalis OMVs give rise to the impediment of lung epithelial cells. We investigated the effect of the OMVs on cell viability and tight junctions of lung epithelial cells. DESIGN: Human lung epithelial A549 cells were treated with P. gingivalis OMVs. Cell viability was evaluated, and cell morphology was examined using scanning electron and phase contrast microscopies. To detect apoptosis induced by P. gingivalis OMVs, activation of caspase-3 and poly ADP-ribose polymerase (PARP) cleavage was examined by using Western blotting. Immunocytochemistry was performed to stain tight junction proteins. RESULTS: P. gingivalis OMVs decreased cell viability in A549 cells in a dose- and time-dependent manner. Microscopic analysis revealed that the OMVs induced morphological changes leading to irregular cell membrane structures. The OMVs caused cell shrinkage, membrane blebbing, and cytoplasmic expulsion in a dose-dependent manner. Western blot analysis showed the OMVs induced caspase-3 activation and PARP cleavage. Treatment with the OMVs disrupted the intact distributions of tight junction proteins. CONCLUSIONS: These results indicate that P. gingivalis OMVs induced cell death by destroying the barrier system in lung epithelial cells. Our present study raises the possibility that P. gingivalis OMVs is an important factor in the engagement of periodontitis with respiratory system diseases.

DOI： 10.1016/j.archoralbio.2020.104841

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium. Reviewed International journal

Jiajie Guo, Ziyi Wang, Yao Weng, Haoze Yuan, Kaya Yoshida, Mika Ikegame, Kenta Uchibe, Hiroshi Kamioka, Kazuhiko Ochiai, Hirohiko Okamura, Lihong Qiu

Cellular signalling 75 109740 - 109740 2020.8

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

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca2+]i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca2+]i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca2+]i mobilization using several extraction methods. The spatial distribution pattern of [Ca2+]i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca2+]i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca2+]i changes. Thus, AHL acts as a double-edged sword for osteoblast function.

DOI： 10.1016/j.cellsig.2020.109740

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver. Reviewed International coauthorship

Seyama M, Yoshida K, Yoshida K, Fujiwara N, Ono K, Eguchi T, Kawai H, Guo J, Weng Y, Haoze Y, Uchibe K, Ikegame M, Sasaki A, Nagatsuka H, Okamoto K, Okamura H, Ozaki K.

Biochimica Biophysica Acta-Molecular Basis of Disease 1866 ( 6 ) 165731 2020.6

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver Reviewed

Mariko Seyama, Kaya Yoshida, Kayo Yoshida, Natsumi Fujiwara, Kisho Ono, Takanori Eguchi, Hotaka Kawai, Jiajie Guo, Yao Weng, Yuan Haoze, Kenta Uchibe, Mika Ikegame, Akira Sasaki, Hitoshi Nagatsuka, Kuniaki Okamoto, Hirohiko Okamura, Kazumi Ozaki

Biochimica et Biophysica Acta - Molecular Basis of Disease 1866 ( 6 ) 2020.6

Publishing type：Research paper (scientific journal)

© 2020 Elsevier B.V. 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

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer.

Ono K, Sogawa C, Kawai H, Tran HK, Taha EA, Lu Y, Oo MW, Okusha Y, Okamura H, Ibaragi S, Takigawa M, Kozaki K, Nagatsuka H, Sasaki A, Okamoto K, Calderwood SK, Eguchi T.

Journal of Extracellular Vesicles 9 ( 1 ) 1769373 2020.5

Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor Reviewed International coauthorship

Okusha Y, Eguchi T, Tran MT, Sogawa C, Yoshida K, Itagaki M, Taha EA, Ono K, Aoyama E, Okamura H, Kozaki KI, Calderwood SK, Takigawa M, Okamoto K.

Cancers (Basel) 12 ( 4 ) E881 2020.4

Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer Reviewed

Yuka Okusha, Takanori Eguchi, Manh T. Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman A. Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

Cancers 12 ( 4 ) 881 - 881 2020.4

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

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

Extracellular Oncosomes Rich in Moonlighting Metalloproteinase (MMP3) Are Transmissive, Pro-Tumorigenic, and Induces Cellular Communication Network Factor 2 (CCN2/CTGF): CRISPR against Cancer

Yuka Okusha, Takanori Eguchi, Manh Tien Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman Ahmed Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

Preprints doi: 10.20944/preprints202002.0281.v1 2020.2

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

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer

Kisho Ono, Chiharu Sogawa, Hotaka Kawai, Manh Tien Tran, Eman A. Taha, Yanyin Lu, May Wathone Oo, Yuka Okusha, Hirohiko Okamura, Soichiro Ibaragi, Masaharu Takigawa, Ken-Ichi Kozaki, Hitoshi Nagatsuka, Akira Sasaki, Kuniaki Okamoto, Stuart K. Calderwood, Takanori Eguchi

Journal of Extracellular Vesicles 9 ( 1 ) 1769373 - 1769373 2020.1

Quorum-sensing molecule N-(3-Oxododecanoyl)-L-Homoserine lactone: An all-rounder in mammalian cell modification. Invited Reviewed International coauthorship

Guo J, Yoshida K, Ikegame M, Okamura H.

Journal of Oral Biosciences 62 ( 1 ) 16 - 29 2020.1

Inhibitory effect of retinoic acid receptor agonists on in vitro chondrogenic differentiation. Reviewed

Sumitani Y, Uchibe K, Yoshida K, Weng Y, Guo J, Yuan H, Ikegame M, Kamioka H, Okamura H

Anatomical Science International 2019

Porphyromonas gingivalis感染したマクロファージが産生する膜小胞が肝臓糖代謝に及ぼす影響 Reviewed

吉田賀弥, 藤原奈津美, 尾崎和美, 内部健太, 池亀美華, 岡村裕彦

Journal of Oral Biosciences Supplement 2018 153 - 153 2018.9

Expression of Noncollagenous Bone Matrix Proteins in Osteoblasts Stimulated by Mechanical Stretching in the Cranial Suture of Neonatal Mice. Reviewed

Ikegame M, Ejiri S, Okamura H

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 22155418793588 2018.8

Reduction of protein phosphatase 2A Cα promotes in vivo bone formation and adipocyte differentiation. Reviewed International journal

Kaya Yoshida, Jumpei Teramachi, Kenta Uchibe, Mika Ikegame, Lihong Qiu, Di Yang, Hirohiko Okamura

Molecular and cellular endocrinology 470 251 - 258 2018.7

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

Serine/threonine protein phosphatase 2A (PP2A) regulates diverse physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Previously, we demonstrated that silencing of the α-isoform of PP2A catalytic subunit (PP2A Cα) in osteoblasts accelerated osteoblast differentiation, whereas its overexpression suppressed differentiation. In this study, we examined the role of PP2A Cα in in vivo bone formation by generating transgenic mice (PP2A-Tg), in which the dominant negative form of PP2A Cα was specifically expressed in osteoblasts. PP2A-Tg mice exhibited an increase in body weight, cortical bone mineral density, and cortical bone thickness. Interestingly, they also displayed higher amounts of adipose tissue in the bone marrow of tibiae. The co-culture study showed that PP2A Cα-knockdown osteoblasts stimulated adipocyte differentiation from undifferentiated mesenchymal cells via upregulation of the adipocyte marker genes, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). These results indicated that the reduction of PP2A Cα levels in osteoblasts promoted bone formation in vivo. Additionally, PP2A Cα in osteoblasts was also potentially involved in controlling adipocyte differentiation through a paracrine mechanism.

DOI： 10.1016/j.mce.2017.11.005

Upregulated osterix expression elicited by Runx2 and Dlx5 is required for the accelerated osteoblast differentiation in PP2A Cα-knockdown cells Reviewed

Di Yang, Hirohiko Okamura, Lihong Qiu

Cell Biology International 42 ( 4 ) 403 - 410 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley-Blackwell Publishing Ltd

Serine/threonine protein phosphatase 2A (PP2A) is involved in regulating various physiological processes including cell cycle, growth, apoptosis, and signal transduction. Osteoblast differentiation is controlled by main bone specific transcription factors including Osterix, distal-less homeobox 5 (Dlx5), and Runt-related transcription factor 2 (Runx2). We previously reported that knockdown of PP2A Cα increases the expression of Osterix, leading to the accelerated osteoblast differentiation through the upregulation of bone-related genes. In this study, we examined whether Dlx5 and Runx2 are involved in the upregulated Osterix expression in PP2A Cα-knockdown osteoblasts (shPP2A cells). The expression of Dlx5 as well as Osterix was significantly higher in shPP2A cells in the initial stage of osteoblast differentiation compared with the control cells (shCont). The expression of Runx2 protein was also higher in shPP2A cells compared with shCont cells although its mRNA level was comparable. Reduction of Dlx5 or Runx2 decreased Osterix expression and alkaline phosphatase activity in shPP2A cells. Luciferase assay showed that Osterix promoter activity was drastically elevated in shPP2A cells compared with that in shCont cells. The deletion or mutation of the Dlx5 and Runx2 binding sites significantly suppressed Osterix promoter activity in shPP2A cells. These results indicate that Dlx5 and Runx2 are critical factors for the upregulated Osterix expression in shPP2A cells, which is considered to be important for the accelerated osteoblast differentiation in these cells.

DOI： 10.1002/cbin.10902

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment Reviewed

Takanori Eguchi, Chiharu Sogawa, Yuka Okusha, Kenta Uchibe, Ryosuke Iinuma, Kisho Ono, Keisuke Nakano, Jun Murakami, Manabu Itoh, Kazuya Arai, Toshifumi Fujiwara, Yuri Namba, Yoshiki Murata, Kazumi Ohyama, Manami Shimomura, Hirohiko Okamura, Masaharu Takigawa, Tetsuya Nakatsura, Ken-ichi Kozaki, Kuniaki Okamoto, Stuart K. Calderwood

PLoS ONE 13 ( 2 ) e0191109 2018.2

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

多彩な役割をもつ蛋白質脱リン酸化酵素PP2A Invited Reviewed

岡村裕彦

岡山歯学会雑誌 36 ( 2 ) 25 - 36 2018

PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts Reviewed

Kaya Yoshida, Hirohiko Okamura, Yuka Hiroshima, Kaori Abe, Jun-ichi Kido, Yasuo Shinohara, Kazumi Ozaki

Experimental Cell Research Vol.354 ( No.1 ) 57 - 64 2017.5

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

The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.

DOI： 10.1016/j.yexcr.2017.03.028

PKR induces the expression of NLRP3 by regulating the NF-kappa B pathway in Porphyromonas gingivalis-infected osteoblasts Reviewed

Kaya Yoshida, Hirohiko Okamura, Yuka Hiroshima, Kaori Abe, Jun-ichi Kido, Yasuo Shinohara, Kazumi Ozaki

EXPERIMENTAL CELL RESEARCH 354 ( 1 ) 57 - 64 2017.5

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

The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-kappa B signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-kappa B activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-kappa B and also induced NLRP3 expression in osteoblasts. Inhibition of NF-kappa B attenuated SNAP -P. g. induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-kappa B and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-kappa B. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-kappa B pathway in periodontal diseases.

DOI： 10.1016/j.yexcr.2017.03.028

PKR regulates LPS-induced osteoclast formation and bone destruction in vitro and in vivo Reviewed

J. Teramachi, Y. Inagaki, H. Shinohara, H. Okamura, D. Yang, K. Ochiai, R. Baba, H. Morimoto, T. Nagata, T. Haneji

ORAL DISEASES 23 ( 2 ) 181 - 188 2017.3

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

ObjectiveIn this study, we aimed to clarify the precise mechanism underlying lipopolysaccharide (LPS)-induced osteoclastogenesis in periodontal disease with a special reference to double-stranded RNA-dependent protein kinase (PKR).
Material and MethodsWe dissected the role of PKR in LPS-induced osteoclast differentiation and function using primary mouse bone marrow cells and RAW264.7 pre-osteoclastic cell line. We used a rat experimental periodontitis (PD) model induced by ligature placement with a Porphyromonas gingivalis LPS injection (PD rat) and analyzed the therapeutic effects of C16, a PKR inhibitor, on bone loss in PD rats.
ResultsProtein kinase is strongly upregulated and phosphorylated by LPS in the osteoclasts. The inhibition of PKR suppressed LPS-stimulated osteoclast formation and activation. PKR inhibition also suppressed the LPS-mediated activation of NF-B and MAPK, which are critical pathways for osteoclastogenesis. High expressions of PKR were detected in osteoclasts of PD rats, and the treatment with C16 effectively prevented alveolar bone destruction in PD rats.
ConclusionsPKR plays a pivotal role in LPS-induced bone loss in PD and, thus, has potential as a therapeutic target for PD.

DOI： 10.1111/odi.12592

Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function Reviewed

Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Jumpei Teramachi, Kazuhiko Ochiai, Tatsuji Haneji, Akihito Yamamoto

JOURNAL OF CLINICAL MEDICINE 6 ( 3 ) 2017.3

Language：English Publisher：MDPI AG

The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/ threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes.

DOI： 10.3390/jcm6030023

Involvement of interleukin-23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis Reviewed

Nan Ma, Di Yang, Hirohiko Okamura, Jumpei Teramachi, Tomokazu Hasegawa, Lihong Qiu, Tatsuji Haneji

MOLECULAR MEDICINE REPORTS 15 ( 2 ) 559 - 566 2017.2

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

Periapical lesions are characterized by the destruction of periapical bone, and occur as a result of local inflammatory responses to root canal infection by microorganisms including Porphyromonas endodontalis (P. endodontalis). P. endodontalis and its primary virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical lesions and alveolar bone loss. Interleukin-23 (IL-23) is critical in the initiation and progression of periodontal disease via effects on peripheral bone metabolism. The present study investigated the expression of IL-23 in tissue where a periapical lesion was present, and the effect of P. endodontalis LPS on the expression of IL-23 in periodontal ligament (PDL) cells. Reverse transcription- quantitative polymerase chain reaction and immunohistochemistry revealed increased levels of IL-23 expression in tissue with periapical lesions compared with healthy PDL tissue. Treatment with P. endodontalis LPS increased the expression of IL-23 in the SH-9 human PDL cell line. BAY11-7082, a nuclear factor kappa B inhibitor, suppressed P. endodontalis LPS-induced IL-23 expression in SH-9 cells. Treatment of RAW264.7 cells with conditioned medium from P. endodontalis LPS-treated SH-9 cells promoted osteoclastogenesis. By contrast, RAW264.7 cells treated with conditioned medium from IL-23-knockdown SH-9 cells underwent reduced levels of osteoclastogenesis. The results of the present study indicated that the expression of IL-23 in PDL cells induced by P. endodontalis LPS treatment may be involved in the progression of periapical lesions via stimulation of the osteoclastogenesis process.

DOI： 10.3892/mmr.2016.6041

Protein phosphatase 2A C alpha regulates proliferation, migration, and metastasis of osteosarcoma cells Reviewed

Di Yang, Hirohiko Okamura, Hiroyuki Morimoto, Jumpei Teramachi, Tatsuji Haneji

LABORATORY INVESTIGATION 96 ( 10 ) 1050 - 1062 2016.10

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

Osteosarcoma is the most frequent primary bone tumor. Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes, such as cell cycle, growth, apoptosis, and signal transduction. In this study, we examined the expression and function of PP2A C alpha in osteosarcoma cells. PP2A C alpha expression was expected to be higher in malignant osteosarcoma tissues. PP2A C alpha expression level and PP2A activity was higher in malignant osteosarcoma LM8 cells compared with that in primary osteoblasts and in the osteoblast-like cell line MC3T3-E1. Okadaic acid, an inhibitor of PP2A, reduced cell viability and induced apoptosis in LM8 cells. PP2A C alpha-knockdown LM8 cells (shPP2A) exhibited less striking filopodial and lamellipodial structures than that in original LM8 cells. Focal adhesion kinase phosphorylation and NF-kappa B activity decreased in shPP2A-treated cells. Sensitivity to serum deprivation-induced apoptosis increased in shPP2A-treated cells, accompanied by a lower expression level of anti-apoptotic BCL-2 in these cells. Reduction of PP2A C alpha resulted in a decrease in the migration ability of LM8 cells in vitro. Reduction in PP2A C alpha levels in vivo suppressed proliferation and metastasis in LM8 cells. PP2A C alpha expression was also higher in human osteosarcoma MG63 and SaOS-2 cells than that in primary osteoblasts and MC3T3-E1 cells, and reduction in PP2A C alpha levels suppressed the cell proliferation rate and migration ability of MG63 cells. These results indicate that PP2A C alpha has a critical role in the proliferation and metastasis of osteosarcoma cells; therefore, its inhibition could potentially suppress the malignancy of osteosarcoma cells.

DOI： 10.1038/labinvest.2016.82

Porphyromonas gingivalis attenuates the insulin-induced phosphorylation and translocation of forkhead box protein O1 in human hepatocytes Reviewed

Haruna Takamura, Kaya Yoshida, Hirohiko Okamura, Natsumi Fujiwara, Kazumi Ozaki

ARCHIVES OF ORAL BIOLOGY 69 19 - 24 2016.9

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

Objective: Porphyromonas gingivalis (P. gingivalis) is a pathogen involved in periodontal disease. Recently, periodontal disease has been demonstrated to increase the risk of developing diabetes mellitus, although the molecular mechanism is not fully understood. Forkhead box protein O1 (FoxO1) is a transcriptional factor that regulates gluconeogenesis in the liver. Gluconeogenesis is a key process in the induction of diabetes mellitus; however, little is known regarding the relationship between periodontal disease and gluconeogenesis. In this study, to investigate whether periodontal disease influences hepatic gluconeogenesis, we examined the effects of P. gingivalis on the phosphorylation and translocation of FoxO1 in insulin-induced human hepatocytes.
Design: The human hepatocyte HepG2 was treated with insulin and Akt and FoxO1 phosphorylation was detected by western blot analysis. The localization of phosphorylated FoxO1 was detected by immunocytochemistry and western blot analysis. HepG2 cells were treated with SNAP26b-tagged P. gingivalis (SNAP-P.g.) before insulin stimulation, and then the changes in Akt and FoxO1 were determined by western blot analysis and immunocytochemistry.
Results: Insulin (100 nM) induced FoxO1 phosphorylation 60 min after treatment in HepG2 cells. Phosphorylated FoxO1 translocated to the cytoplasm. SNAP-P.g. internalized into HepG2 cells and decreased Akt and FoxO1 phosphorylation induced by insulin. The effect of insulin on FoxO1 translocation was also attenuated by SNAP-P.g.
Conclusions: Our study shows that P. gingivalis decreases the phosphorylation and translocation of FoxO induced by insulin in HepG2 cells. Our results suggest that periodontal disease may increase hepatic gluconeogenesis by reducing the effects of insulin on FoxO1. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2016.05.010

Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim. Reviewed International journal

Di Yang, Hirohiko Okamura, Jumpei Teramachi, Tatsuji Haneji

Biochimica et biophysica acta 1863 ( 4 ) 650 - 9 2016.4

Language：English

Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.

DOI： 10.1016/j.bbamcr.2016.01.006

Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim Reviewed

Di Yang, Hirohiko Okamura, Jumpei Teramachi, Tatsuji Haneji

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1863 ( 4 ) 650 - 659 2016.4

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

Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2016.01.006

PKR regulates LPS-induced osteoclast formation and bone destruction in vitro and in vivo. Reviewed

Jumpei Teramachi, Yuji Inagaki, Hiroki Shinohara, Hirohiko Okamura, Di Yang, Kazuhiko Ochiai, Ryoko Baba, Hiroyuki Morimoto, Toshihiko Nagata, Tatsuji Haneji

Oral Diseases 2016

Histone Demethylase Utx Regulates Differentiation and Mineralization in Osteoblasts Reviewed

Di Yang, Hirohiko Okamura, Jumpei Teramachi, Tatsuji Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 116 ( 11 ) 2628 - 2636 2015.11

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

Alteration of methylation status of lysine 27 on histone H3 (H3K27) associates with dramatic changes in gene expression in response to various differentiation signals. Demethylation of H3K27 is controlled by specific histone demethylases including ubiquitously transcribed tetratricopeptide repeat X chromosome (Utx). However, the role of Utx in osteoblast differentiation remains unknown. In this study, we examined whether Utx should be involved in osteoblast differentiation. Expression of Utx increased during osteoblast differentiation in MC3T3-E1 cells and primary osteoblasts. GSK-J1, a potent inhibitor of H3K27 demethylase, increased the levels of trimethylated H3K27 (H3K27me3) and decreased the expressions of Runx2 and Osterix and ALP activity in MC3T3-E1 cells. Stable knockdown of Utx by shRNA attenuated osteoblast differentiation and decreased ALP activity, calcium content, and bone-related gene expressions. Silencing of Utx increased the level of H3K27me3 on the promoter regions of Runx2 and Osterix and decreased the promoter activities of Runx2 and Osterix. Taken together, our present results propose that Utx plays important roles in osteoblast differentiation by controlling the expressions of Runx2 and Osterix. J. Cell. Biochem. 116: 2628-2636, 2015. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25210

Double Stranded RNA-Dependent Protein Kinase is Necessary for TNF--Induced Osteoclast Formation In Vitro and In Vivo Reviewed

Hiroki Shinohara, Jumpei Teramachi, Hirohiko Okamura, Di Yang, Toshihiko Nagata, Tatsuji Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 116 ( 9 ) 1957 - 1967 2015.9

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

Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF- plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF--induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF--induced osteoclastogenesis. The TNF--induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pretreatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K, and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF--induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF--induced activation of NF-B and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-B and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF--induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF--induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF--induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF--induced osteoclast differentiation in vitro and in vivo. J. Cell. Biochem. 116: 1957-1967, 2015. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25151

Preventive Effect of Daiokanzoto (TJ-84) on 5-Fluorouracil-Induced Human Gingival Cell Death through the Inhibition of Reactive Oxygen Species Production Reviewed

Kaya Yoshida, Masami Yoshioka, Hirohiko Okamura, Satomi Moriyama, Kazuyoshi Kawazoe, Daniel Grenier, Daisuke Hinode

PLOS ONE 9 ( 11 ) e112689 2014.11

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

Daiokanzoto (TJ-84) is a traditional Japanese herbal medicine (Kampo formulation). While many Kampo formulations have been reported to regulate inflammation and immune responses in oral mucosa, there is no evidence to show that TJ-84 has beneficial effects on oral mucositis, a disease resulting from increased cell death induced by chemotherapeutic agents such as 5-fluorouracil (5-FU). In order to develop effective new therapeutic strategies for treating oral mucositis, we investigated (i) the mechanisms by which 5-FU induces the death of human gingival cells and (ii) the effects of TJ-84 on biological events induced by 5-FU. 5-FU-induced lactate dehydrogenase (LDH) release and pore formation in gingival cells (Sa3 cell line) resulted in cell death. Incubating the cells with 5-FU increased the expression of nucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) and caspase-1. The cleavage of caspase-1 was observed in 5-FU-treated cells, which was followed by an increased secretion of interleukin (IL)-1 beta. The inhibition of the NLRP3 pathway slightly decreased the effects of 5-FU on cell viability and LDH release, suggesting that NLRP3 may be in part involved in 5-FU-induced cell death. TJ-84 decreased 5-FU-induced LDH release and cell death and also significantly inhibited the depolarization of mitochondria and the up-regulation of 5-FU-induced reactive oxygen species (ROS) and nitric oxide (NO) production. The transcriptional factor, nuclear factor-kappa B (NF-kappa B) was not involved in the 5-FU-induced cell death in Sa3 cells. In conclusion, we provide evidence suggesting that the increase of ROS production in mitochondria, rather than NLRP3 activation, was considered to be associated with the cell death induced by 5-FU. The results also suggested that TJ-84 may attenuate 5-FU-induced cell death through the inhibition of mitochondrial ROS production.

DOI： 10.1371/journal.pone.0112689

Reduction of PP2A Cα stimulates adipogenesis by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression. Reviewed International journal

Hirohiko Okamura, Di Yang, Kaya Yoshida, Jumpei Teramachi, Tatsuji Haneji

Biochimica et biophysica acta 1843 ( 11 ) 2376 - 84 2014.11

Language：English

Serine/threonine protein phosphatase 2A (PP2A) regulates several physiological processes such as the cell cycle, cell growth, apoptosis, and signal transduction. In this study, we examined the expression and role of PP2A Cα in adipocyte differentiation. PP2A Cα expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPARγ and adiponectin increased. To further clarify the role of PP2A Cα in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A Cα (shPP2A cells). Silencing of PP2A Cα in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A Cα suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3β (phospho-GSK-3β), leading to the reduction of β-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3β inhibitor, and inhibition of PPARγ expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A Cα plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression.

DOI： 10.1016/j.bbamcr.2014.06.008

Reduction of PP2A C alpha stimulates adipogenesis by regulating the Wnt/GSK-3 beta/beta-catenin pathway and PPAR gamma expression Reviewed

Hirohiko Okamura, Di Yang, Kaya Yoshida, Jumpei Teramachi, Tatsuji Haneji

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1843 ( 11 ) 2376 - 2384 2014.11

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

Serine/threonine protein phosphatase 2A (PP2A) regulates several physiological processes such as the cell cycle, cell growth, apoptosis, and signal transduction. In this study, we examined the expression and role of PP2A C alpha in adipocyte differentiation. PP2A C alpha expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPAR gamma and adiponectin increased. To further clarify the role of PP2A C alpha in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A C alpha (shPP2A cells). Silencing of PP2A C alpha in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A C alpha suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3 beta (phospho-GSK-3 beta), leading to the reduction of beta-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3 beta inhibitor, and inhibition of PPAR gamma expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A C alpha plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3 beta/beta-catenin pathway and PPAR gamma expression. (C) 2014 Published by Elsevier B.V.

DOI： 10.1016/j.bbamcr.2014.06.008

Tumor necrosis factor-alpha induces interleukin-34 expression through nuclear factor-kappa B activation in MC3T3-E1 osteoblastic cells Reviewed

Yaqiong Yu, Di Yang, Lihong Qiu, Hirohiko Okamura, Jiajie Guo, Tatsuji Haneji

MOLECULAR MEDICINE REPORTS 10 ( 3 ) 1371 - 1376 2014.9

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

Osteoblasts produce various types of cytokines under pathological conditions and control osteoclast differentiation. Tumor necrosis factor-alpha (TNF-alpha) has been demonstrated to exert complex effects in osteoblasts under local inflammatory conditions, including in periodontal and periapical diseases. Interleukin-34 (IL-34) has been recently identified as a novel regulatory factor for the differentiation and function of osteoclasts. The present study provides the first evidence, to the best of our knowledge, that the expression of IL-34 is induced by TNF-alpha through nuclear factor-kappa B (NF-kappa B) activation in MC3T3-E1 osteoblastic cells. TNF-alpha induced IL-34 expression in a dose- and time-dependent manner. Immunocytochemistry with an NF-kappa B antibody demonstrated that NF-kappa B was mainly localized in the cytoplasm of the untreated MC3T3-E1 cells. Rapid translocation of NF-kappa B from the cytoplasm to the nucleus was observed in the cells treated with TNF-alpha for 15 min. Translocation and transcriptional activity of NF-kappa B were also determined by western blotting and a luciferase reporter assay, respectively. Pretreatment with 100 mu M CAPE, an inhibitor of NF-kappa B, significantly inhibited TNF-alpha-induced IL-34 expression. These results indicate that TNF-alpha induces IL-34 expression via NF-kappa B in osteoblasts.

DOI： 10.3892/mmr.2014.2353

Calcium Hydroxide Suppresses Porphyromonas endodontalis Lipopolysaccharide-induced Bone Destruction Reviewed

J. Guo, D. Yang, H. Okamura, J. Teramachi, K. Ochiai, L. Qiu, T. Haneji

JOURNAL OF DENTAL RESEARCH 93 ( 5 ) 508 - 513 2014.5

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS INC

Porphyromonas endodontalis and its main virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical diseases and alveolar bone loss. Calcium hydroxide is commonly used for endodontic therapy. However, the effects of calcium hydroxide on the virulence of P. endodontalis LPS and the mechanism of P. endodontalis LPS-induced bone destruction are not clear. Calcium hydroxide rescued the P. endodontalis LPS-suppressed viability of MC3T3-E1 cells and activity of nuclear factor-kappa B (NF-kappa B) in these cells, resulting in the reduced expression of interleukin-6 and tumor necrosis factor-alpha. In addition, calcium hydroxide inhibited P. endodontalis LPS-induced osteoclastogenesis by decreasing the activities of NF-kappa B, p38, and ERK1/2 and the expression of nuclear factor of activated T-cell cytoplasmic 1 in RAW264.7 cells. Calcium hydroxide also rescued the P. endodontalis LPS-induced osteoclastogenesis and bone destruction in mouse calvaria. Taken together, our present results indicate that calcium hydroxide suppressed bone destruction by attenuating the virulence of P. endodontalis LPS on bone cells.

DOI： 10.1177/0022034514526886

Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3β signaling pathway. Reviewed International journal

Makoto Ishikawa, Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Haruna Takamura, Natsumi Fujiwara, Kazumi Ozaki

Biochimica et biophysica acta 1832 ( 12 ) 2035 - 43 2013.12

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

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.

DOI： 10.1016/j.bbadis.2013.07.012

Oral porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3b signaling pathway Reviewed

Ishikawa Makoto, Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Takamura Haruna, Natsumi Fujiwara, Kazumi Ozaki

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease Vol.1832 ( No.12 ) 2035 - 2043 2013.12

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

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.

DOI： 10.1016/j.bbadis.2013.07.012

Histone Demethylase Jmjd3 Regulates Osteoblast Differentiation via Transcription Factors Runx2 and Osterix Reviewed

Di Yang, Hirohiko Okamura, Yoshiki Nakashima, Tatsuji Haneji

JOURNAL OF BIOLOGICAL CHEMISTRY 288 ( 47 ) 33530 - 33541 2013.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Post-translational modifications of histones including methylation play important roles in cell differentiation. Jumonji domain-containing 3 (Jmjd3) is a histone demethylase, which specifically catalyzes the removal of trimethylation of histone H3 at lysine 27 (H3K27me3). In this study, we examined the expression of Jmjd3 in osteoblasts and its roles in osteoblast differentiation. Jmjd3 expression in the nucleus was induced in response to the stimulation of osteoblast differentiation as well as treatment of bone morphogenetic protein-2 (BMP-2). Either treatment with Noggin, an inhibitor of BMP-2, or silencing of Smad1/5 suppressed Jmjd3 expression during osteoblast differentiation. Silencing of Jmjd3 expression suppressed osteoblast differentiation through the expression of bone-related genes including Runx2, osterix, osteopontin, bone sialoprotein (BSP), and osteocalcin (OCN). Silencing of Jmjd3 decreased the promoter activities of Runx2 and osterix and increased the level of H3K27me3 on the promoter regions of Runx2 and osterix. Introduction of the exogenous Runx2 and osterix partly rescued osteoblast differentiation in the shJmjd3 cells. The present results indicate that Jmjd3 plays important roles in osteoblast differentiation and regulates the expressions of BSP and OCN via transcription factors Runx2 and osterix.

DOI： 10.1074/jbc.M113.497040

Protein phosphatase 2A C alpha regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor Reviewed

Hirohiko Okamura, Kaya Yoshida, Di Yang, Tatsuji Haneji

JOURNAL OF CELLULAR PHYSIOLOGY 228 ( 5 ) 1031 - 1037 2013.5

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

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in a-isoform of PP2A catalytic subunit (PP2A Ca) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Ca in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Ca. Overexpression of PP2A Ca reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Ca-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Ca-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Ca-overexpressing cells. These results indicate that PP2A Ca and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Ca-mediated osteoblast differentiation. J. Cell. Physiol. (C) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/jcp.24250

Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor. Reviewed

Hirohiko Okamura, Kaya Yoshida, Di Yang, Tatsuji Haneji

Journal of Cellular Physiology Vol.228 ( No.5 ) 1031 - 1037 2013.5

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

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation.

DOI： 10.1002/jcp.24250

Protein phosphatase 2A C is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts. Reviewed

Hirohiko Okamura, Di Yang, Kaya Yoshida, Tatsuji Haneji

FEBS Letters Vol.587 ( No.1 ) 48 - 53 2013.1

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

We examined whether alteration of PP2A Cα expression in osteoblasts is involved in osteoclast differentiation. Reduction of PP2A Cα in MC3T3-E1 cells (shPP2A) decreased receptor activator of nuclear factor κB ligand (RANKL) expression and increased osteoprotegerin (OPG) expression. The conditioned medium from shPP2A cells failed to induce NFATc1 as well as the expression of osteoclast marker genes cathepsin K and osteoclast-associated receptor (OSCAR) in bone marrow macrophage cells. Treatment of bone marrow macrophage cells with the conditioned medium from shPP2A cells impaired osteoclastogenesis. These results suggest that alteration of PP2A Cα expression in osteoblasts modulates the expressions of RANKL and OPG, which are involved in osteoclastogenesis via the NFATc1 transcription factor.

DOI： 10.1016/j.febslet.2012.10.041

Protein phosphatase 2A C alpha is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts Reviewed

Hirohiko Okamura, Di Yang, Kaya Yoshida, Tatsuji Haneji

FEBS LETTERS 587 ( 1 ) 48 - 53 2013.1

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

We examined whether alteration of PP2A C alpha expression in osteoblasts is involved in osteoclast differentiation. Reduction of PP2A C alpha in MC3T3-E1 cells (shPP2A) decreased receptor activator of nuclear factor kappa B ligand (RANKL) expression and increased osteoprotegerin (OPG) expression. The conditioned medium from shPP2A cells failed to induce NFATc1 as well as the expression of osteoclast marker genes cathepsin K and osteoclast-associated receptor (OSCAR) in bone marrow macrophage cells. Treatment of bone marrow macrophage cells with the conditioned medium from shPP2A cells impaired osteoclastogenesis. These results suggest that alteration of PP2A C alpha expression in osteoblasts modulates the expressions of RANKL and OPG, which are involved in osteoclastogenesis via the NFATc1 transcription factor. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2012.10.041

PKR plays a positive role in osteoblast differentiation by regulating GSK-3β activity through a β-catenin-independent pathway Reviewed

Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Yumi Hoshino, Tatsuji Haneji, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

Molecular and Cellular Endocrinology 361 ( 1-2 ) 99 - 105 2012.9

Publishing type：Research paper (scientific journal)

Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3β (GSK-3β) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3β inhibitor, increased GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3β phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a β-catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3β activity through a β-catenin-independent pathway. © 2012 Elsevier Ireland Ltd.

DOI： 10.1016/j.mce.2012.03.019

PKR plays a positive role in osteoblast differentiation by regulating GSK-3b activity through a b-catenin-independent pathway Reviewed

Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Yumi Hoshimo, Tatsuji Haneji, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

Molecular and Cellular Endocrinology Vol.361 ( No.1-2 ) 99 - 105 2012.9

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

Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3 (GSK-3) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3 inhibitor, increased GSK-3 phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3 phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3 phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a -catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3 activity through a -catenin-independent pathway.

DOI： 10.1016/j.mce.2012.03.019

Interaction between PKR and PACT mediated by LPS-inducible NF-?B in human gingival cells Reviewed

Kaya Yoshida, Hirohiko Okamura, Yumi Hoshino, Masayuki Shono, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

JOURNAL OF CELLULAR BIOCHEMISTRY 113 ( 1 ) 165 - 173 2012.1

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

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 mu g/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-?B to the nucleus after 30 min, and inhibition of NF-B decreased the PACTPKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFa), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-?B decreased it. These results indicated that LPS induces PKR phosphorylation and the PACTPKR association in Sa3 cells. Our results also suggest that NF-?B is involved in the PACTPKR interaction and the production of pro-inflammatory cytokines in periodontitis. J. Cell. Biochem. 113: 165173, 2012. (C) 2011 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.23340

Interaction between PKR and PACT mediated by LPS-inducible NF-kB in human gingival cells Reviewed

Kaya Yoshida, Hirohiko Okamura, Yumi Hoshimo, Masayuki Shono, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

Journal of Cellular Biochemistry Vol.113 165 - 173 2012.1

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

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 μg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-κB to the nucleus after 30 min, and inhibition of NF-κB decreased the PACT-PKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT-PKR association in Sa3 cells. Our results also suggest that NF-κB is involved in the PACT-PKR interaction and the production of pro-inflammatory cytokines in periodontitis.

DOI： 10.1002/jcb.23340

Reduction of protein phosphatase 2A C alpha enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix Reviewed

Hirohiko Okamura, Kaya Yoshida, Kazuhiko Ochiai, Tatsuji Haneji

BONE 49 ( 3 ) 368 - 375 2011.9

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

The serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as control of cell cycle, growth, and division. On the other hand, Osterix is a zinc-finger-containing transcription factor that is essential for the differentiation of osteoblasts and regulation of many bone-related genes. Here we examined the effect of okadaic acid (OA), a specific inhibitor of PP2A, on bone formation in vivo and the molecular mechanism regulated by PP2A C alpha in osteoblast differentiation. Administration of 1 nM OA to the calvarial region in mice increased bone mineral density, as shown by mu CT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1 nM OA stimulated osteoblast differentiation and the expression of Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN) in mouse osteoblastic MC3T3-E1 cells. Moreover, the expression and phosphatase activity of PP2A C alpha was decreased in the initial step of osteoblast differentiation, which was in parallel with an increase in Osterix expression. To further clarify the role of PP2A C alpha in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A C alpha. Accordingly, the silencing of PP2A Cot in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, lisp, and OCN. Our data indicate that PP2A C alpha plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2011.06.004

Reduction of protein phosphatase 2A Cα enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix. Reviewed

Hirohiko Okamura, Kaya Yoshida, Kazuhiko Ochiai, Tatsuji Haneji

Bone Vol.49 ( No.3 ) 368 - 375 2011.6

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

The serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as control of cell cycle, growth, and division. On the other hand, Osterix is a zinc-finger-containing transcription factor that is essential for the differentiation of osteoblasts and regulation of many bone-related genes. Here we examined the effect of okadaic acid (OA), a specific inhibitor of PP2A, on bone formation in vivo and the molecular mechanism regulated by PP2A Cα in osteoblast differentiation. Administration of 1nM OA to the calvarial region in mice increased bone mineral density, as shown by μCT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1nM OA stimulated osteoblast differentiation and the expression of Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN) in mouse osteoblastic MC3T3-E1 cells. Moreover, the expression and phosphatase activity of PP2A Cα was decreased in the initial step of osteoblast differentiation, which was in parallel with an increase in Osterix expression. To further clarify the role of PP2A Cα in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A Cα. Accordingly, the silencing of PP2A Cα in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, Bsp, and OCN. Our data indicate that PP2A Cα plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes.

DOI： 10.1016/j.bone.2011.06.004

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells Reviewed

Heni Susilowati, Hirohiko Okamura, Katsuhiko Hirota, Masayuki Shono, Kaya Yoshida, Keiji Murakami, Atsushi Tabata, Hideaki Nagamune, Tatsuji Haneji, Yoichiro Miyake

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 404 ( 1 ) 57 - 61 2011.1

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

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-kappa B translocation in human hepatic HepG2 cells, ILY did not affect NF-kappa B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.11.057

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells. Reviewed

Heni Susilowati, Hirohiko Okamura, Katsuhiko Hirota, Masayuki Shono, Kaya Yoshida, Keiji Murakami, Atsushi Tabata, Hideaki Nagamune, Tatsuji Haneji, Yoichiro Miyake

Biochemical and Biophysical Research Communications Vol.404 ( No.1 ) 57 - 61 2011

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

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

DOI： 10.1016/j.bbrc.2010.11.057

Negative regulation of TIMP1 is mediated by transcription factor TWIST1 Reviewed

Hirohiko Okamura, Kaya Yoshida, Tatsuji Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 35 ( 1 ) 181 - 186 2009.7

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

TWIST1 is involved in tumor invasion and metastasis by promoting epithelial-to-mesenchymal transition. However, the molecular target of TWIST1 involving this mechanism is poorly understood. To identify the TWIST-regulated genes, we established the Saos-2 cells stably expressing TWIST1 gene by transfecting TWIST1 cDNA into the cells and performed a differential display approach by using annealing control primers. Here, we report that one of the genes that were down-regulated in TWIST1 expressing cells is predicted to be TIMP1. TIMP1 has been reported as the naturally occurring specific inhibitor of matrix metalloproteinases (MMPs). Overexpression of TWIST1 gene suppressed the expression of TIMP1 mRNA but had no effect on TIMP2 and MMP-2 expression, as determined by semi-quantitative RT-PCR. We showed that TWIST1 was up-regulated in SCCBHY cells, which have a strong capacity of invasion into mandibular bone compared with SCCHN cells. Our present results suggest that TWIST1 is involved in tumor invasion by regulating the expression of TIMP1.

DOI： 10.3892/ijo_00000327

PKR-mediated degradation of STAT1 regulates osteoblast differentiation Reviewed

Kaya Yoshida, Hirohiko Okamura, Bruna Rabelo Amorim, Daisuke Hinode, Hideo Yoshida, Tatsuji Haneji

EXPERIMENTAL CELL RESEARCH 315 ( 12 ) 2105 - 2114 2009.7

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

The double-stranded RNA-dependent protein kinase (PI(R) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I Collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2009.02.003

PKR-mediated degradation of STAT1 regulates osteoblast differentiation Reviewed

Kaya Yoshida, Hirohiko Okamura, Bruna Rabelo Amorim, Daisuke Hinode, Hideo Yoshida, Tatsuji Haneji

Experimental Cell Research Vol.315 ( No.12 ) 2105 - 2114 2009.7

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

The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.

DOI： 10.1016/j.yexcr.2009.02.003

Calcineurin regulates phosphorylation status of transcription factor osterix Reviewed

Hirohiko Okamura, Bruna Rabelo Amorim, Jie Wang, Kaya Yoshida, Tatsuji Haneji

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379 ( 2 ) 440 - 444 2009.2

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

Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix Such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical Study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.12.094

Calcineurin regulates phosphorylation satus of transcriptionfactor osterix Reviewed

Hirohiko Okamura, Bruna Rabelo Amorim, Jie Wang, Kaya Yoshida, Tatsuji Haneji

Biochemical and Biophysical Research Communications Vol.379 ( No.2 ) 440 - 444 2009

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

Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.

DOI： 10.1016/j.bbrc.2008.12.094

Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells Reviewed

Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Tatsuji Haneji

Journal of Cellular Biochemistry Vol.103 ( No.5 ) 1488 - 1496 2008.4

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

Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin.

DOI： 10.1002/jcb.21537

Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells Reviewed

Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Tatsuji Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 103 ( 5 ) 1488 - 1496 2008.4

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

Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 mu M bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin.

DOI： 10.1002/jcb.21537

The transcriptional factor Osterix directly interacts with RNA helicase A. Invited Reviewed

Amorim BR, Okamura H, Yoshida K, Wang J, Qiu LH, Haneji T

Ocean Papers & Printers, New Delhi 216 - 222 2008

Cobalt chloride induces apoptosis and zinc chloride suppresses cobalt-induced apoptosis by Bcl-2 expression in human submandibular gland HSG cells Reviewed

Kazumi Akita, Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Hiroaki Ogawa-Iyehara, Tatsuji Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 31 ( 4 ) 923 - 929 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

To determine the effects of cobalt chloride on human submandibular gland cells, HSG cells were exposed to various concentrations of cobalt chloride. Cobalt chloride induced cytotoxicity and cell death in HSG cells as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin were observed in cobalt chloride-treated cells. Cobalt chloride induced DNA ladder formation in HSG cells in both dose- and time-dependent manner with maximal effect at a concentration of 0.5 mM and 48 h, respectively. Cobalt chloride inhibited the expression of both Bcl-2 protein and mRNA in dose- and time-dependent manner. Zinc chloride recovered the cobalt-suppressed Bcl-2 expression and protected against cobalt-induced apoptosis in HSG cells. Our results show that the pathway of the apoptosis in HSG cells is regulated by cobalt chloride and zinc chloride. Our results also indicate that cobalt-induced apoptotic steps in HSG cells are related to the production of Bcl-2 protein.

Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-kappa B in human osteoblastic osteosarcoma MG63 cells Reviewed

Hiroaki Tanaka, Kaya Yoshida, Hirohiko Okamura, Hiroyuki Morimoto, Toshihiko Nagata, Tatsuji Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 31 ( 2 ) 389 - 396 2007.8

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappa B could be affected by the treatment with calyculin A. In this report, we demonstrate that calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappa B, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with antiphospho-p65NF-kappa B antibody disclosed that the NF-kappa B was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-kappa B was further confirmed by using the phosphorylation site-mutated p65NF-kappa B gene transfected into HEK293 cells. Unlike TNF-alpha, calyculin A treatment did not degraded I kappa B alpha within 10 min, while it degraded I kappa B alpha at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-kappa B on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappa B to the nucleus, thereby promoting transcriptional activity of NF-kappa B-related genes.

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest Reviewed

Hiroyuki Morimoto, Akiko Ozaki, Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Hiroaki Tanaka, Seiichiro Kitamura, Tatsuji Haneji

CELL BIOCHEMISTRY AND FUNCTION 25 ( 4 ) 369 - 375 2007.7

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

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PPI) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PPI isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PPIs and nucleolin was not changed. Anti-nucleolin antibody interacted with 1 10-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G2/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PPI and nucleolin were differently expressed at G(1)/S and G2/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression. Copyright (C) 2005 John Wiley & Sons, Ltd.

DOI： 10.1027/cbf.1300

Calyculin A stimulates the expression of TNF-alpha mRNA via phosphorylation of Akt in mouse osteoblastic MC3T3-E1 cells Reviewed

Lihong Qiu, Kaya Yoshida, Bruna Rabelo Amorim, Hirohiko Okamura, Tatsuji Haneji

MOLECULAR AND CELLULAR ENDOCRINOLOGY 271 ( 1-2 ) 38 - 44 2007.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-alpha mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that calyculin A increased the expression of TNF-alpha mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the calyculin A-stimulated TNF-alpha mRNA expression. Western blot result disclosed that calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and S8203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on calyculin A-stimulated expression of TNF-alpha mRNA. BAY11-7085 and CAPE, inhibitors of NF-kappa B activity, did not alter the calyculin A-stimulated TNF-alpha mRNA expression. Indirect immunofluorescent study confirmed that NF-kappa B was not translocated to the nucleus by calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-alpha mRNA. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mce.2007.03.005

The transcriptional factor Osterix directly interacts with RNA helicase A Reviewed

Bruna Rabelo Amorim, Hirohiko Okamura, Kaya Yoshida, Lihong Qiu, Hiroyuki Morimoto, Tatsuji Haneji

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 355 ( 2 ) 347 - 351 2007.4

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

Osterix is an osteoblast-specific transcriptional factor, required for bone formation and osteoblast differentiation. Here, we identified new Osterix interacting factors by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the candidates, RNA helicase A was identified to interact with Osterix. To determine the interaction of Osterix with RNA helicase A, immunoprecipitation assay was performed. Western analysis confirmed the association between Osterix and RNA helicase A. Immunocytochemical analysis also showed that Osterix and RNA helicase A were co-localized in HEK 293 cells. Our data suggest that RNA helicase A might be a component of Osterix regulation. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.01.150

Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells Reviewed

Hirohiko Okamura, Kaya Yoshida, Eiko Sasaki, Lihong Qiu, Bruna Rabelo Amorim, Hiroyuki Morimoto, Tatsuji Haneji

CELL BIOLOGY INTERNATIONAL 31 ( 2 ) 119 - 125 2007.2

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

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphomositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 mu g/ml, 0-6 h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.cellbi.2006.09.014

口腔扁平上皮癌細胞の薬剤耐性とアポトーシス ~ 転写調節因子の役割 ~ Reviewed

岡村裕彦

四国歯学会雑誌 19 171 - 17 2007

Role of transcription factors in multidrug resistance and apoptosis. Invited Reviewed

Okamura H, Haneji T

Dentistry in Japan 43 ( 1 ) 1 - 6 2007

Okadaic acid induces phosphorylation of p65NF-kappa B on serine 536 and activates NF-kappa B transcriptional activity in human osteoblastic MG63 tells Reviewed

Akiko Ozaki, Hiroyuki Morimoto, Hiroaki Tanaka, Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Tatsuji Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 99 ( 5 ) 1275 - 1284 2006.12

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

Nuclear factor-kappa B (NF-kappa B) is an essential. transcription factor in the control of expression of genes involved in coil growth, differentiation, inflammation, and neoplastic transformation. Previously, we reported that okadaic acid (OA), which is a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in cells of human osteosarcoma cell line MG63. However, to date, it is hot clear whether the phosphorylation status of NF-kappa B could be affected by the treatment with OA. in this report, we demonstrate that treatment of MG63 cells with OA enhanced the phosphorylation level of NF-kappa B, as judged from the results of Western blot analysis and a protein phosphatase dephosphorylation assay. The phosphorylation level of NF-kappa B was enhanced in both time- and dose-dependent manners. In the cells treated with 100nM OA for 3 h, consequential translocation of NF-kappa B from the cytosol to the nucleus occurred. Western blotting experiments with an anti-phospho-p65NF-kappa B antibody disclosed that the NF-kappa B was phosphorylated on serine 536. Furthermore, OA stimulated the transcriptional activity of NF-kappa B in MG63 cells, as judged from the results of a luciferase assay. Our findings indicate that OA elicit phosphorylation of NF-kappa B on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappa B to the nucleus, thereby promoting transcriptional activity of genes.

DOI： 10.1002/jcb.20873

Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro Reviewed

K Yoshida, H Okamura, BR Amorim, A Ozaki, H Tanaka, H Morimoto, T Haneji

EXPERIMENTAL CELL RESEARCH 311 ( 1 ) 117 - 125 2005.11

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

In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1 alpha(STAT1 alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse ostcoblastic MC3T3-E1 cells, thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1 alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1 alpha a protein in PKR Mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1 alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1 alpha and/or Runx2 expression. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2005.09.006

Okadaic acid induces tyrosine phosphorylation of I kappa B alpha that mediated by PKR pathway in human osteoblastic MG63 cells Reviewed

H Morimoto, A Ozaki, H Okamura, K Yoshida, S Kitamura, T Haneji

MOLECULAR AND CELLULAR BIOCHEMISTRY 276 ( 1-2 ) 211 - 217 2005.8

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

Treatment of human osteosarcoma cell line MG63 cells with okadaic acid stimulated phosphorylation of I kappa B alpha, as judged from the results of Western blot analysis and a lambda protein phosphatase dephosphorylation assay. The stimulated phosphorylation of I kappa B alpha was both time- and dose-dependent. The phosphorylation sites of I kappa B alpha were taken to be tyrosine residues because the anti-phospho-tyrosine antibody bound to the samples immunoprecipitated with the anti-I kappa B alpha antibody. In the cells treated with 100 nM okadaic acid consequential translocation of NF-kappa B p65 from the cytosol to the nucleus occurred. Double-stranded RNA-dependent protein kinase (PKR) is a player in the cellular antiviral response and is involved in transcriptional stimulation through activation of NF-kappa B. We investigated the functional relationship between PKR and I kappa B alpha phosphorylation by constructing MG63 PKR K/R cells that produced a catalytically inactive mutant PKR. NF-kappa B p65 was detected in the nucleus of these cells, even in the unstimulated cells. Although I kappa B alpha was degraded phosphorylation of eIF-2 alpha, a substrate of PKR, did not occur in the mutant cells treated with okadaic acid. Our results suggest that okadaic acid-induced tyrosine phosphorylation of I kappa B alpha was mediated by PKR kinase activity, thus indicating the involvement of this kinase in the control mechanism governing the activation of NF-kappa B.

DOI： 10.1007/s11010-005-4440-y

PTEN expression elicited by EGR-1 transcription factor in calyculin a-induced apoptotic cells Reviewed

H Okamura, K Yoshida, H Morimoto, T Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 94 ( 1 ) 117 - 125 2005.1

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

PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3-kinase-Akt pathway. The gene for transcription factor EGR-1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR-1 and the phosphorylation status of EGR-1 and Akt in calyculin A-treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR-1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with calyculin A in time- and dose-dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the calyculin A-treated SCCTF cells. Calyculin A-stimulated expression of FGR-1 and PTEN might be p53 independent, because the expression of them was also detected in p53-null Saos-2 cells. RNA interference using double-stranded RNA specific for the EGR-1 gene inhibited not only EGR-1 expression but also PTEN expression in SCCTF cells treated or not with calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by calyculin A. These observations also support the view that EGR-1 regulates PTEN expression in the initial steps of the apoptotic pathway. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcb.20283

Okadaic acid induces apoptosis through double-stranded RNA-dependent protein kinase/eukaryotic initiation factor-2 alpha pathway in human osteoblastic MG63 cells Reviewed

H Morimoto, H Okamura, K Yoshida, S Kitamura, T Haneji

JOURNAL OF BIOCHEMISTRY 136 ( 4 ) 433 - 438 2004.10

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

Double-stranded RNA-dependent protein kinase (PKR) is a participant in the cellular antiviral response and phosphorylates the a-subunit of eukaryotic translation initiation factor 2alpha (eIF-2alpha) to block protein synthesis. Treatment of human osteosarcoma cell line MG63 cells with a serine and threonine protein phosphatase inhibitor, okadaic acid, at the concentration of 100 nM, but not at 20 nM, induced apoptosis. To investigate the functional relationship between phosphatases and apoptosis, we examined the phosphorylation levels of PKR and eIF-2a by Western blot analysis. During treatment of cells with it at the higher concentration (100 nM), okadaic acid increased the level of phosphorylated PKR in MG63 cells, this kinase phosphorylating eIF-2alpha. However, at the lower concentration (20 nM), okadaic acid did not affect the level of phosphorylated PKR. In the cells treated with 100 nM okadaic acid, activation of NF-kappaB also occurred. Even though inhibition of translation occurred simultaneously in MG63 cells, the expression of pro-apoptotic proteins Fas and Bax was not affected by 100 nM okadaic acid in these cells. We concluded that the inhibition of translation decreased anti-apoptotic protein expression, thus resulting in apoptosis. Our results also suggest that the inhibition of the protein phosphatase activity by okadaic acid induced apoptosis in MG63 cells through PKR and eIF-2alpha.

DOI： 10.1093/jb/mvh144

Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells Reviewed

H Okamura, K Yoshida, E Sasaki, H Morimoto, T Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 25 ( 4 ) 1031 - 1037 2004.10

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated. RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells. With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells. Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel. Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells. The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells. The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdrl promoter activity. NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells. Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells.

Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells Reviewed

H Morimoto, H Okamura, K Yoshida, S Kitamura, T Hanejl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 19 ( 4 ) 327 - 331 2004.8

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of alpha and gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.

DOI： 10.1080/14756360410001704452

Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells Reviewed

M Fujita, K Goto, K Yoshida, H Okamura, H Morimoto, S Kito, J Fukuda, T Haneji

ORAL ONCOLOGY 40 ( 2 ) 199 - 206 2004.2

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

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility get shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(03)00152-0

薬剤耐性扁平上皮癌細胞におけるアポトーシスと転写調節因子 Reviewed

岡村裕彦

徳島大学大学院歯学研究科 17 ( 1 ) 123 - 136 2004

Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells Reviewed

E Sasaki, Y Okamoto, K Yoshida, H Okamura, K Shimizu, F Nasu, H Morimoto, T Haneji

HISTOCHEMISTRY AND CELL BIOLOGY 120 ( 4 ) 327 - 333 2003.10

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

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.

DOI： 10.1007/s00418-003-0572-x

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells Reviewed

S Kito, K Shimizu, H Okamura, K Yoshida, H Morimoto, M Fujita, Y Morimoto, T Ohba, T Haneji

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 ( 4 ) 950 - 956 2003.1

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

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02942-X

Okadaic acid stimulates the receptor activator of nuclear factor-kappa B ligand (RANKL) in mouse osteoblastic cells. Reviewed

Yoshida K, Okamura H, Morimoto H, Nagata T, Haneji T

Biomed. Res. 14 ( 2 ) 126 - 132 2003

Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid Reviewed

H Okamura, H Morimoto, M Fujita, F Nasu, E Sasaki, T Haneji

ORAL ONCOLOGY 38 ( 8 ) 779 - 784 2002.12

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

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type I and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(02)00039-8

Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid Reviewed

H. Okamura, H. Morimoto, M. Fujita, F. Nasu, E. Sasaki, T. Haneji

Oral Oncology 38 ( 8 ) 779 - 784 2002.12

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

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level. © 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(02)00039-8

Interaction of protein phosphatase 1 delta with nucleolin in human osteoblastic cells Reviewed

H Morimoto, H Okamura, T Haneji

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 50 ( 9 ) 1187 - 1193 2002.9

Language：English Publishing type：Research paper (scientific journal) Publisher：HISTOCHEMICAL SOC INC

We examined the expression and cytolocalization of the protein phosphatase type 1 delta (PP1delta) isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Cellular fractionation of MG63 cells was done and protein was prepared from each fraction. Anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole-cell lysate. The 100-kD protein was detected in nuclear and nucleolar fractions. The 95-kD protein was detected in cytosolic and nucleoplasmic fractions. PP1delta and nucleolin were co-localized in the nucleolus in MG63 and Saos-2 cells revealed by an immunofluorescence method. PP1delta and nucleolin were also co-immunoprecipitated with anti-nucleolin and anti-PP1delta antibodies. In the actinomycin D-treated cells, the subcellular localization of PP1delta and nucleolin was changed. Expression of PP1delta was upregulated with actinomycin D treatment. The level of 100-kD protein did not change in the actinomycin D-treated cells. However, the level of the 95-kD band increased with actinomycin D treatment. These results indicate that PP1delta was associated with nucleolin in the nucleolus of MG63 and Saos-2 cells and that nucleolin is a possible candidate substrate for PP1delta.

DOI： 10.1177/002215540205000905

Peplomycin-induced apoptosis in oral squamous carcinoma cells depends on bleomycin sensitivity Reviewed

H Okamura, H Morimoto, T Haneji

ORAL ONCOLOGY 37 ( 4 ) 379 - 385 2001.6

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

Oral squamous carcinoma cell line SSCKN cells were shown to be highly sensitive to bleomycin, whereas SCCTF cells were minimally sensitive to this reagent. To determine whether the anticancer drug resistance to oral squamous carcinoma cells could be related to the degree of the drug-induced apoptosis, we examined the effects of peplomycin on induction of apoptosis in these cells. After reaching subconfluence, SCCKN and SCCTF cells were exposed to various concentrations of peplomycin. Peplomycin caused cytotoxicity in both SCCKN and SCCTF cells in a dose-dependent fashion with the maximal effect at concentrations of 1 and 10 muM, respectively, as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, we observed marked nuclear condensation and fragmentation of chromatin in SCCKN cells treated with 1 muM peplomycin. How ever, SCCTF cells treated with 1 muM peplomycin showed neither nuclear condensation nor fragmentation. DNA ladder formation was also detected in both cell lines by treatment with peplomycin. The induced DNA ladder formation in SCCKN and SCCTF cells was dose-dependent, with the maximal effect at concentrations of 5 and 50 muM, respectively. Bleomycin also induced DNA ladder formation in SCCKN and SCCTF cells with different sensitivities. Mitomycin C induced DNA laddering in both SCCKN and SCCTF cells; however, the intensity of DNA ladder formation was almost the same in both cell lines. The present results indicate that peplomycin-induced apoptosis in oral squamous carcinoma cell lines depends on the sensitivity of these cells to bleomycin. (C) 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(00)00101-9

Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cells Reviewed

Y Morimoto, S Kito, T Ohba, H Morimoto, H Okamura, T Haneji

JOURNAL OF ORAL PATHOLOGY & MEDICINE 30 ( 4 ) 193 - 199 2001.4

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

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 hDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein, in the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis.

DOI： 10.1034/j.1600-0714.2001.300401.x

Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid Reviewed

Y Morimoto, H Morimoto, H Okamura, K Nomiyama, N Nakamuta, S Kobayashi, S Kito, T Ohba, T Haneji

ARCHIVES OF ORAL BIOLOGY 45 ( 8 ) 657 - 666 2000.8

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

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells, The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells. (C) 2000 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0003-9969(00)00038-8

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