Faculty Profiles - NAKAYAMA Masaaki

The fungal metabolite (+)-terrein abrogates osteoclast differentiation via suppression of the RANKL signaling pathway through NFATc1. International journal

International immunopharmacology 83 106429 - 106429 2020.6

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

DOI： 10.1016/j.intimp.2020.106429

PCRをベースとしたLautropia mirabilisの簡便な検出法と分離法の検討

佐藤あやめ, 中山真彰, 中川美緒, 小崎弘貴, 室美里, 曽我賢彦, 大原直也

日本細菌学雑誌 75 ( 1 ) 66 - 66 2020.1

Construction and characterization of a PGN_0297 mutant of Porphyromonas gingivalis: evidence for contribution of PGN_0297 to gingipain activity Reviewed

Shintaro Ono, Masaaki Nakayama, Masato Tachibana, Abu Saleh Muhammad Shahriar, Wang Heling, Shogo Takashiba, and Naoya Ohara

Acta Medica Okayama 2019.3

Construction and characterization of a PGN_0297 mutant of Porphyromonas gingivalis: Evidence of the contribution of PGN_0297 to gingipain activity

Shintaro Ono, Masaaki Nakayama, Masato Tachibana, Abu Saleh Muhammad Shahriar, Wang Heling, Shogo Takashiba, Naoya Ohara

Acta Medica Okayama 73 ( 4 ) 315 - 323 2019

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

The periodontal pathogen Porphyromonas gingivalis shows colonial pigmentation on blood agar and produces gingipains (Kgp, RgpA, and RgpB), cysteine proteases involved in an organism's virulence and pigmentation. We showed previously that deletion of the PGN_0300 gene abolished the pigmentation activity and reduced the proteolytic activity of gingipains. The role of the PGN_0297 gene, which consists of an operon with the PGN_0300 gene, is unclear. Herein we examined the effect of PGN_0297 gene deletion on the pigmentation and proteolytic activities and transcriptional levels of gingipains. A PGN_0297 gene deletion mutant (ΔPGN_0297) did not exhibit the pigmentation. The proteolytic activity of the gingipains was decreased in the culture supernatant and on the cell surface of ΔPGN_0297. The mutant ΔPGN_0297 failed to attenuate Akt phosphorylation at Thr308 and Ser473, but both phosphorylations were attenuated in the wild-type and its complementation strain. The deletion of PGN_0297 gene did not substantially affect the transcriptional levels of the gingipain genes kgp, rgpA, and rgpB. Taken together, these results indicate that PGN_0297 is closely involved in the secretion and maturation of gingipains.

Fungal metabolite (+)-terrein suppresses IL-6/sIL-6R-induced CSF1 secretion by inhibiting JAK1 phosphorylation in human gingival fibroblasts. Reviewed International journal

Satoshi Yamamoto, Kazuhiro Omori, Hiroki Mandai, Masaaki Nakayama, Saki Nakagawa, Hiroya Kobayashi, Tadashi Kunimine, Hiroshi Yoshimura, Kyosuke Sakaida, Hidefumi Sako, Soichiro Ibaragi, Tadashi Yamamoto, Hiroshi Maeda, Seiji Suga, Shogo Takashiba

Heliyon 4 ( 11 ) e00979 2018.11

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

Control of bacterial infection-induced inflammatory responses is one of the effective therapeutic approaches of periodontal diseases. Natural products such as lipid mediators and metabolites from microorganisms have been used for decreasing inflammation. We previously reported that (+)-terrein inhibited activation of STAT3 and ERK1/2 in interleukin-6 (IL-6) signaling cascade, leading to prevent vascular endothelial growth factor (VEGF) secretion in human gingival fibroblasts (HGFs). However, little is still known about the role of (+)-terrein on inflammatory responses. In this study, we provided the possibility of novel action that (+)-terrein inhibits activation of Janus-activated kinase 1 (JAK1), which has a central function in IL-6 signaling cascade, and alters expression of mRNAs and proteins induced by IL-6/soluble IL-6 receptor (sIL-6R) stimulation in HGFs. First, we performed PCR array to examine IL-6/sIL-6R-induced mRNA expression, and then expression of mRNA and protein of colony stimulating factor-1 (CSF1) and VEGF were clearly determined by quantitative RT-PCR and ELISA, respectively. Treatment with (+)-terrein suppressed expression of mRNA and protein of CSF1 and VEGF by IL-6/sIL-6R stimulation. Next, to test the effect of (+)-terrein on IL-6/sIL-6R signaling cascade, we demonstrated whether (+)-terrein affects phosphorylation of JAK1 and its downstream proteins, Akt and SHP-2. Western blotting revealed that (+)-terrein inhibited IL-6/sIL-6R-induced phosphorylation of JAK1, Akt, and SHP-2. Therefore, (+)-terrein suppresses IL-6/sIL-6R-induced expression of CSF1 and VEGF via inhibition of JAK1, Akt, and SHP-2. Based on our results, we suggest that (+)-terrein is a candidate compound for anti-inflammatory effect associated with IL-6 signaling.

DOI： 10.1016/j.heliyon.2018.e00979

Fungal metabolite (

HELIYON 4 ( 11 ) 2018.11

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

Control of bacterial infection-induced inflammatory responses is one of the effective therapeutic approaches of periodontal diseases. Natural products such as lipid mediators and metabolites from microorganisms have been used for decreasing inflammation. We previously reported that (+)-terrein inhibited activation of STAT3 and ERK1/2 in interleukin-6 (IL-6) signaling cascade, leading to prevent vascular endothelial growth factor (VEGF) secretion in human gingival fibroblasts (HGFs). However, little is still known about the role of (+)-terrein on inflammatory responses. In this study, we provided the possibility of novel action that (+)-terrein inhibits activation of Janus-activated kinase 1 (JAK1), which has a central function in IL-6 signaling cascade, and alters expression of mRNAs and proteins induced by IL-6/soluble IL-6 receptor (sIL6R) stimulation in HGFs. First, we performed PCR array to examine IL-6/sIL6R-induced mRNA expression, and then expression of mRNA and protein of colony stimulating factor-1 (CSF1) and VEGF were clearly determined by quantitative RT-PCR and ELISA, respectively. Treatment with (+)-terrein suppressed expression of mRNA and protein of CSF1 and VEGF by IL-6/sIL-6R stimulation. Next, to test the effect of (+)-terrein on IL-6/sIL-6R signaling cascade, we demonstrated whether (+)-terrein affects phosphorylation of JAK1 and its downstream proteins, Akt and SHP-2. Western blotting revealed that (+)-terrein inhibited IL-6/sIL-6R-induced phosphorylation of JAK1, Akt, and SHP-2. Therefore, (+)-terrein suppresses IL-6/sIL-6R-induced expression of CSF1 and VEGF via inhibition of JAK1, Akt, and SHP-2. Based on our results, we suggest that (+)-terrein is a candidate compound for anti-inflammatory effect associated with IL-6 signaling.

DOI： 10.1016/j.heliyon.2018.e00979

Novel function of Porphyromonas gingivalis gingipains in the PI3K/Akt signaling pathway Invited Reviewed

Nakayama Masaaki, Ohara Naoya

JOURNAL OF ORAL BIOSCIENCES 59 ( 3 ) 131 - 134 2017.8

Language：English Publisher：Japanese Association for Oral Biology

Background Porphyromonas gingivalis is s major oral bacterium closely associated with periodontal diseases including periodontitis and directly affects host cellular signaling. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays multiple roles in various cell functions including cell survival and glucose metabolism. In this review, we describe the effect of gingipains on the PI3K/Akt signaling pathway in P. gingivalis infection. Highlight Gingipains inactivate PI3K and Akt in gingival epithelial cells infected with P. gingivalis. These events occur independently of invasion of this organism into the cells and are required for the enzymatic activity of gingipains. Furthermore, 3-Phosphoinositide-dependent protein kinase-1 (PDK1) failed to translocate to the plasma membrane from the cytosol following PI3K inactivation. Additionally, dephosphorylation of Akt downstream proteins, including glycogen synthase kinase 3 (GSK3), mammalian target of rapamycin (mTOR), and Bad, occurs in parallel with the dysregulation of PI3K/PDK1/Akt cascades. Conclusion This review describes the biological characterization of gingipains, which inactivate PI3K and Akt, and disorder the PI3K/Akt signaling pathway. Hence, gingipains may decrease cellular physiological functions, eventually disrupting the gingival epithelium and causing development of periodontal diseases.

DOI： 10.1016/j.job.2017.05.003

Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis Reviewed

Yuko Taguchi, Keiko Sato, Hideharu Yukitake, Tetsuyoshi Inoue, Masaaki Nakayama, Mariko Naito, Yoshio Kondo, Konami Kano, Tomonori Hoshino, Koji Nakayama, Shogo Takashiba, Naoya Ohara

INFECTION AND IMMUNITY 84 ( 1 ) 230 - 240 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

DOI： 10.1128/IAI.01308-15

BCG thyX欠損株の作製とその性状解析

有村友紀, 中山真彰, 妹尾昌紀, 田川淳平, 中山浩次, 飯田征二, 大原直也

Journal of Oral Biosciences Supplement 2015 330 - 330 2015.9

BCGにおけるcyclic-di-GMP菌体内合成の影響

妹尾昌紀, 中山真彰, 有村友紀, 飯田征二, 大原直也

Journal of Oral Biosciences Supplement 2015 331 - 331 2015.9

Involvement of an Skp-like protein, PGN_0300, in the type IX secretion system of Porphyromonas gingivalis Reviewed

Yuko Taguchi , Keiko Sato , Hideharu Yukitake , Tetsuyoshi Inoue , Masaaki Nakayama , Mariko Naito , Yoshio Kondo , Konami Kano , Tomonori Hoshino , Koji Nakayama , Shogo Takashiba and Naoya Ohara

Infection and Immunity 290 ( 8 ) 5190 - 5202 2015.2

Attenuation of the phosphatidylinositol 3-Kinase/Akt signaling pathway by porphyromonas gingivalis gingipains RgpA, RgpB, and Kgp

Masaaki Nakayama, Tetsuyoshi Inoue, Mariko Naito, Koji Nakayama, Naoya Ohara

Journal of Biological Chemistry 290 ( 8 ) 5190 - 5202 2015.2

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Biochemistry and Molecular Biology Inc.

Background: The significance of gingipains on the PI3K/Akt signaling pathway is poorly understood.Results: The inactivation of PI3K and Akt was induced by P. gingivalis, which was associated with gingipains.Conclusion: Gingipains are critical for suppressing the PI3K/Akt signaling pathway via extracellular proteolysis independent of P. gingivalis invasion. Significance: This study shows the first evidence that the gingipains negatively regulate the PI3K/Akt signaling pathway.

DOI： 10.1074/jbc.M114.591610

Attenuation of the PI3 kinase/Akt signaling pathway by Porphyromonas gingivalis gingipains RgpA, RgpB, and Kgp Reviewed

Masaaki Nakayama, Tetsuyoshi Inoue, Mariko Naito, Koji Nakayama and Naoya Ohara

The Journal of Biological Chemistry 290 ( 8 ) 5190 - 5202 2015.1

Characterization of the tripartite drug efflux pumps of Porphyromonas gingivalis ATCC 33277 Reviewed

Tetsuyoshi Inoue, Masaaki Nakayama, Yuko Taguchi, Konami Kano, Miyuu Ono, Yurika Shimizu, Teruo Kuroda, Naoya Ohara

New Microbiologica 38 ( 1 ) 97 - 103 2015.1

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis. Reviewed International journal

Satosu Onozawa, Yuichiro Kikuchi, Kazuko Shibayama, Eitoyo Kokubu, Masaaki Nakayama, Tetsuyoshi Inoue, Keisuke Nakano, Yukinaga Shibata, Naoya Ohara, Koji Nakayama, Kazuyuki Ishihara, Toshiyuki Kawakami, Hiromasa Hasegawa

BMC oral health 15 4 - 4 2015.1

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

BACKGROUND: Porphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation. METHODS: To elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining. RESULTS: Comparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation. CONCLUSION: These results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

DOI： 10.1186/1472-6831-15-4

Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis Reviewed

Junpei Tagawa, Tetsuyoshi Inoue, Mariko Naito, Keiko Sato, Tomomi Kuwahara, Masaaki Nakayama, Koji Nakayama, Takashi Yamashiro, Naoya Ohara

JOURNAL OF MICROBIOLOGICAL METHODS 105 174 - 179 2014.10

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

We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-Bacteroides/P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5 kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mimet.2014.07.032

Hemoglobin Receptor Protein from Porphyromonas gingivalis Induces Interleukin-8 Production in Human Gingival Epithelial Cells through Stimulation of the Mitogen-Activated Protein Kinase and NF-κB Signal Transduction Pathways Reviewed

Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis Reviewed

Junpei Tagawa, Tetsuyoshi Inoue, Mariko Naito, Keiko Sato, Tomomi Kuwahara, Masaaki Nakayama, Koji Nakayama, Takashi Yamashiro, Naoya Ohara

Journal of Microbiological Methods 2014.8

Yuki Fujita, Masaaki Nakayama, Mariko Naito, Eiki Yamachika, Tetsuyoshi Inoue, Koji Nakayama, Seiji Iida, Naoya Ohara

Infection and Immunity 82 ( 1 ) 202 - 211 2014.1

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

ABSTRACT

Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobic bacterium Porphyromonas gingivalis , which is implicated as an important pathogen for chronic periodontitis, triggers a series of host inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis , hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles of HbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the human gingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blocked HbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-κB-p65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA (siRNA) targeting activating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 production. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, respectively. Combined pretreatment with an inhibitor of NF-κB (BAY11-7082) and SB203580 was more efficient in inhibiting the ability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells, the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-κB, thus resulting in the induction of IL-8 production.

DOI： 10.1128/iai.01140-12