Kyudai Oral Bioscience 2017
(KOB2017)
Physiological and Pathological Roles of
Lysosomal Proteolytic System

February 11th (Sat) 2017
Collaboration Station I, 2F Audiovisual Hall
Kyushu University

PROGRAM & ABSTRACTS

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Information for Speakers■ Presentation Instruments
Presentations are restricted to computer presentations using your own
personal computer.
Please use a computer running Windows XP, Windows Vista, Windows 7-10,
Machintosh OS X or later and fitted with an external monitor output
terminal.
All speakers are also requested to bring the data of your presentation on aUSB. Please mark your name and the session number (e.g. S1-1) on the file.
Please make sure that virus check is executed beforehand.

■ Making Presentations
For the speakers in Special Lecture, please bring your own personal
computer to the Computer Operating Desk at 12:40.
All speakers are requested to operate the computer by themselves.
Your cooperation in finishing your presentation within the allotted time isappreciated.
After your presentation, please reclaim your computer from the Computer
Operating Desk.

2

PROGRAM3

Kyudai Oral Bioscience 2017 (KOB2017)
13:0013:10
Hiroshi Nakanishi(Representative Organizer of KOB)

Opening Remarks
Masato Hirata(Dean, Faculty of Dental Science, Kyushu University)

Special Lectures
Physiological and Pathological Roles of Lysosomal Proteolytic System
Chair person Masato Hirata(Laboratory of Molecular and Cellular Biochemistry)

13:1014:10 Special Lecture 1
Veronika Stoka
Lysosomal cysteine cathepsins and their protein inhibitors, cystatins, in healthand disease
Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, Ljubljana,
Slovenia

Chair persons     

Seiji Nakamura (Section of Oral and Maxillofacial Oncology)
Hiroshi Nakanishi (Department of Aging Science and Pharmacology)

14:2015:00 Special Lecture 2
Masato Koike
The role of autophagy and lysosomal proteolysis for the maintenance of thenormal environment of central nervous system
Department of Cell Biology and Neuroscience, Juntendo University Graduate School of
Medicine, Tokyo, Japan

15:0015:40 Special Lecture 3
Yoichi Ezura
Paradoxical osteoclast functions in congenital osteolytic disorders
Skeletal Molecular Pharmacology, Division of Advanced Molecular Medicine,
Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan4

15:4016:20 Special Lecture 4
Xian-Wu Cheng
Role of cysteinyl protease cathepsins in atherosclerosis-based cardiovasculardisease: Focus on novel biology and mechanisms
Institute of Innovation for Future Society, Nagoya University, and the Department of Community
Health & Geriatrics, Nagoya University Graduate School of Medicine, Nagoya Japan

16:2016:40

Group PhotographPhD Student Session (PSS) Chair personYuka Harada
16:4017:00 Peports of Short-term Exchange Program (JASSO)
Sanako Nakaya, Wakako Nakayama,Tihiro Furumi, Masato Yamamoto
Report after attending Gadjah Mada University dental summer course
School of Dental Science, Kyushu University

17:0017:10 PSS-1
Erika Tomoda
Epigenetic regulation of PITX2 causes dysfunction of stem cells from apical
papilla in dentin dysplasia type I
Departments of Molecular Cell Biology and Oral Anatomy, Faculty of Dental Science, Kyushu
University

17:1017:20 PSS-2
Yoshikazu Hayashi
Uncarboxylated osteocalcin involves antitumor immunity in cancer growth
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu
University

17:2017:30 PSS-3
Yicong Liu
Infection of microglia with Porphyromonas gingivalis promotes cellmigration
and an inflammatory response through the gingipain-mediated activation ofprotease-activated receptor-2
Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University5

17:3017:40 PSS-4
Shinya Kageyama
Exploration of IgA-binding bacteria in salivary microbiome
Section of Preventive and Public Health Dentistry, Faculty of Dental Science, Kyushu University

17:4017:50 Coffee Break
17:5018:00 PSS-5
Masahiko Morioka
Exosomes from oral squamous carcinoma cells define the tropism ofinvasiveness and lymphatic dissemination
Section of Oral and Maxillofacial Oncology, Faculty of Dental Science, Kyushu University

18:0018:10 PSS-6
Mitsudai Tsuruta
The effect of innate micro-inflammation on pancreatic islet cells
Department of Periodontology, Faculty of Dental Science, Kyushu University

18:1018:20 PSS-7
Hiromi Mitarai
Transgelin mediates the proliferation of human periodontal ligament cells
induced by TGF-β1
Department of Endodontology and Operative Dentistry, Faculty of Dental Science, Kyushu
University

18:2018:30 PSS-8
Kota Tsutsumi
Characterisation of sucrose-independent supragingival plaque produced in an invitro biofilm model
Section of Oral Health Promotion and Technology, Graduate School of Dental Science, Kyushu
University

Closing Remarks
18:3018:40
Fusanori Nishimura(Department of Periodontology)

6

ABSTRACTS

7

Special Lecutures
Physiological and Pathological Roles of
Lysosomal Proteolytic System

8Special Lecture 1
Lysosomal cysteine cathepsins and their protein
inhibitors, cystatins, in health and disease
Veronika Stoka
Department of Biochemistry and Molecular and Structural Biology,
J. Stefan Institute, Ljubljana, Slovenia

Lysosomes and endosomes contain proteases, primarily cathepsins, namely, the aspartic
cathepsins D and E (pepsin family) and 11 human cysteine cathepsins, B, C, F, H, K, L, O, S, V, W
and X (papain family). These enzymes differ in their localization and tissue distribution, expression
profiles, biochemical properties, structures and the regulation of their proteolytic activities [1,2].
Cysteine cathepsins are synthesized as inactive precursors and activated at acidic pH into mature
active enzymes. They are all monomeric proteins except the tetrameric cathepsin C. Their
extracellular localization is often bound to an increased expression and activity such as elastinolytic
and collagenolytic activity i.e. cathepsins S, K and V responsible for the remodelling of theextracellular matrix (ECM) [2]. The structures of distinct cathepsins explain their broad substratespecificities. Cathepsin activities can be controlled by various mechanisms such as pH, zymogen
activation, and endogenous protein and exogenous low Mr inhibitors [1].
The endogenous protein inhibitors of the cystatin superfamily have been classified into three
types: the stefins (Family 1), the cystatins (Family 2), and the kininogens (Family 3) [3]. Cystatin
inhibitory activity is essential for the delicate regulation of normal physiological processes bylimiting the potentially harmful activity of their target proteases. Failures in biological mechanisms
controlling protease activities result in many diseases such as neurodegeneration [4,5],
cardiovascular diseases, inflammatory diseases, such as rheumatoid arthritis, and cancer, among
others [1].
There are a few genetic disorders involving mutations in cathepsin genes. Genetic studies
revealed that loss-of-function mutations in the cathepsin C (DPPI) gene results in early-onset

9periodontitis and palmoplantar keratosis, characteristics of Haim-Munk and Papillon-Lefevre
syndromes [6]. In the cathepsin K gene, at least fifteen mutations in humans leading topycnodysostosis, are known [7]. Studies on cysteine cathepsins biology contribute to developmentof new therapies in various diseases with high probability of being incorporated into clinical trials.

References[1] Turk V., Stoka V., Vasiljeva O., Renko M., Sun T., Turk B., Turk D. (2012) Biochim Biophys
Acta. 1824:68-88.
[2] Turk B., Turk D., Turk V.

(2012) EMBO J. 31:1630-43.

[3] Turk V., Stoka V., Turk D. (2008) Front Biosci. 13:5406-20.
[4] Nakanishi H., Wu Z. (2009) Behav Brain Res. 201:1-7.
[5] Stoka V., Turk V., Turk B. (2016) Ageing Res Rev. 32:22-37.
[6] Hewitt C. et al. (2004) Human Mutation 23:222-8. [7] Motyckova G., Fisher D.E. (2002) CurrMol Med. 2:407-21.

10

Special Lecture 2
The role of autophagy and lysosomal proteolysis forthe maintenance of the normal environment ofcentral nervous system
Masato Koike
Department of Cell Biology and Neuroscience, Juntendo University
Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo, 113-8421, Japan

We have been analyzing brain tissues from cathepsin D (CD) deficient mice, a model for themost severe type of neuronal ceroid lipofuscinoses (NCLs). NCLs are a group of inherited,neurodegenerative, lysosomal storage disorders characterized by progressive intellectual and motor
deterioration, seizures, and early death. The group of human neuronal NCLs currently comprises 14
genetically distinct disorders (CLN1-14).
Most types of NCLs are pathologically characterized by storage of autofluorescent material
containing subunit c of mitochondrial ATP synthase (SCMAS) within lysosomes. Mitochondria aresequestrated by autophagy/mitophagy resulting in the degradation by lysosomal proteinases, such as
tripeptidyl peptidase 1 (TPP1)/CLN2 and CD/CLN10. NCLs are classified as rare diseases.
However, taking into account that in many cases of early-onset familial Parkinson's disease (PD),genes essential for mitophagy are mutated, NCL and PD may share etiological features. Indeed,mutations in ATP13A2 are a known cause of Kufor-Rakeb syndrome (KRS) with both PD
phenotypes and NCL pathology. Thus ATP13a2 is called both Park9 and CLN13.
In this symposium I will review our previous studies on the several genetic mouse models for
elucidation of autophagy-lysosomal systems in neurons under physiologic and pathologic
conditions and further introduce about the recent studies on the comparative analyses of CD and
ATP13a2 deficient mice and Purkinje cells-selective CD and Atg7-deficient mice.

11

Special Lecture 3
Paradoxical osteoclast functions in congenital
osteolytic disorders
Yoichi Ezura
Skeletal Molecular Pharmacology, Division of Advanced Molecular Medicine,
Medical Research Institute, Tokyo Medical and Dental University (TMDU)
1-5-45 Yushima Bunkyo-ku, Tokyo, 113-8510 JapanThe osteolysis occurs in various clinical situations such as loosening orthopaedic prosthesis,
infectious dental implants, and metastatic malignant tumors. In addition, various genetic disorders are
known to be associated with osteolysis. In most situations, activated osteoclast function andinflammation would explain the symptoms. However, in some situations, the findings are paradoxical.
For example, an osteopetrotic disorder named Pycnodysostosis by defective homozygous mutation in
Cathepsin K is known to be associated with paradoxical fingertip bone erosion, named as"acro-osteolysis". Similarly, carpal bone osteolysis could be somehow paradoxically caused byhomozygous MMP2 deficiency or by MMP14 deficiency in the patients of “multicentric osteolysis
nodulosis arthropathy (MONA)”. Indeed in our experiment, Mmp2 deficient mice had paradoxical
osteopenia in the long bone metaphysis in contrast to the thickened cortical and calvarial bones,
consistent with the symptoms found in human patients. Although the pathophysiological mechanismsof osteolysis are still need to be understood, we are trying to unveil the relationships between theosteoclast functions and location specific osteolysis in several mutant mouse lines. In this talk, I
would like to talk about the osteolytic and osteopenic phenotypes of the mutant mice that we had
analyzed in the past few years. The relationships between the findings on Dok-deficient mice,Cnot3-hemizygous mice, conditional Profilin-deficient mice and genetic disorders would be
discussed.

12       Special Lecture 4
Role of cysteinyl protease cathepsins inatherosclerosis-based cardiovascular disease:
Focus on novel biology and mechanisms
Xian-Wu Cheng         Institute of Innovation for Future Society, Nagoya University,and the Department of Community Health & Geriatrics,

Nagoya University Graduate School of Medicine, Nagoya Japan

Until recently, the role of lysosomal cysteine protease cathepsins in intracellular protein
degradation was believed to be mainly restricted to scavenging. However, recent studies haverevealed nontraditional roles for cysteine protease cathepsins in the extracellular space during thedevelopment and progression of cardiovascular disease. Although the precise mechanisms are
unknown, data from animal studies suggest that members of the cathepsin family, like otherextracellular proteases, contribute to extracellular matrix protein remodeling and interstitial matrix
degradation, as well as to cell signaling and cell apoptosis in heart disease. Serum levels ofcathepsins L, S, and K and their endogenous inhibitor cystatin C may be useful predictive
biomarkers in patients with coronary artery disease and cardiac disease. Furthermore, in vivo
pharmacological intervention with a synthetic cathepsin inhibitor and cardiovascular drugs has thepotential for pharmacologic targeting of cathepsins in cardiovascular disease. This review focuseson cathepsin biology and the involvement of cysteinyl cathepsins in the pathogenesis of several
heart and vessel diseases, especially with respect to their potential application as diagnostic andprognostic markers and drug targets to prevent inappropriate proteolysis in cardiovascular disease.

13   

PhD Student Session

14

Peports of Short-term Exchange Program (JASSO)

Report after attending Gadjah Mada University dental summer
courseSanako Nakaya, Wakako Nakayama,Tihiro Furumi, Masato Yamamoto
School of Dental Science, Kyushu University, Fukuoka, 812-8582, Japan
We visited Gadjah Mada University in Yogyakarta, Indonesia. It takes about 1 hour by air planefrom the capital city, Jakarta. There are many islands in Indonesia and Yogyakarta is located in Javaisland. The city has many temples like Kyoto. Gadjah Mada University is a national university built
in 1949. It has 18 faculties, for example, law, medicine, engineering and so on. We participated in
UGM dental summer course hosted by the Faculty of Dentistry. We studied with the students fromYonsei University in Korea and Malaya University in Malaysia. From Japan, there were 5
attendants from Kyushu and Tokushima University. The course contained visiting regional hospital,
clinical skill training, language class (English, Javanese), wearing traditional clothes, sightseeing,and so on. Here, we talk about what we experienced, felt, and learned about the dentistry andculture in Indonesia, and would like to give a message to the junior students coming after us.

15

PSS-1
Epigenetic regulation of PITX2 causes dysfunction of stem cells fromapical papilla in dentin dysplasia type I
Erika Tomoda1,2, Haruyoshi Yamaza2, Soichiro Sonoda1, Yosuke Tanaka1,
Norihisa Uehara1, Yukari N. Kyumoto1, Toshio Kukita1, Sontao Shi3, Kazuaki
Nonaka2, Takayoshi Yamaza1
1

Departments of Molecular Cell Biology and Oral Anatomy and 2Pediatric Dentistry, Kyushu
University Graduate School of Dental Science, Fukuoka 812-8582, Japan3
Department of Anatomy and Cell Biology, University of Pennsylvania School of Dental
Medicine, Philadelphia, PA 19104, USA

Tooth root formation is associated with epithelial-mesenchymal interaction between epithelial
sheath of Hertwig and odonotoblasts. Stem Cells from Apical Papilla (SCAP) are responsible
mesenchymal stem cells for forming tooth root dentin. Dentin dysplasia type I (DDI) is an
autosomal-dominant irritant clinically associated with tooth root aplasia and/or hypoplasia.
However, the critical gene and mechanism of DDI have not been elucidated. Recently, we clinically
met with a patient of non-autosomal-dominant DDI non-associated with ectodermal deficiency,
suggesting that the tooth root abnormality might be occurred by dysfunction of SCAP. Cells wereisolated from the apical papilla-like tissue of the patient teeth and characterized as SCAP-like cells,
DDI-SCAP. We then evaluated that DDI-SCAP impaired odontogenic differentiation capacity
associated with dentin sialophosphoprotein and enhanced Runx2 expression when compared withhealthy-donor derived control SCAP (control SCAP). In addition, DDI-SCAP damaged the cell
cycle, especially G1 phase associated with cyclin dependent kinase 6 (CDK6), CDK2 and cyclin D1.
Molecular and biochemical analyses revealed that PITX2 gene and protein was markedly reduced in
DDI-SCAP in comparison with control SCAP. However, the genetic deletion and mutation of
PITX2 were not observed in DDI-SCAP. Furthermore, we evaluated that PITX2 siRNA-knockdown
suppressed odontogenic differentiation and G1 phase of cell cycle in control SCAP, suggesting that
epigenetically regulated PITX2 gene cause the dysfunction of cell differentiation and cell cycle of
SCAP in DDI.
Key words: Dentin Dysplasia Type I (DDI), Stem Cells from Apical Papilla (SCAP), PITX2, Cell
Cycle

16

PSS-2
Uncarboxylated osteocalcin involves antitumor immunity
in cancer growth
Yoshikazu Hayashi1,2, Tomoyo Kawakubo-Yasukochi1,4, Akiko Mizokami1,3,
Seiji Nakamura2, Masato Hirata1
1

Laboratory of Molecular and Cellular Biochemistry, 2Section of Oral and Maxillofacial
Oncology, 3OBT Research Center, Faculty of Dental Science, Kyushu University, Fukuoka
812-8582, Japan, 4Department of Immunological and Molecular Pharmacology, Faculty of
Pharmaceutical Science, Fukuoka University, Fukuoka 814-0180, Japan
Osteocalcin (OC), a noncollagenous bone matrix protein secreted by osteoblasts, in serum has
been correlated with bone remodeling under pathological status including cancer bone metastasis,
as well as normal skeletal turnover. OC in serum exits as two types, γ-carboxylated (GlaOC) or
lower- (or un-)γ-carboxylated (GluOC). Recent studies demonstrated that high circulating OC levels
constitute a marker for bone metastasis in prostate cancer patient. We previously reported that
GluOC potentially suppresses human prostate cancer cell growth by inhibiting receptor tyrosine
kinases (RTKs) activities. However, the mechanisms in vivo have not been elucidated.
In this study, we found that GluOC suppressed tumor growth of B16 mouse melanoma
transplants in C57Bl/6N wild-type mice, but GluOC did not exhibit antitumor activity in humanprostate cancer xenografts using athymic nude mice. Moreover, stimulation of primary mousesplenocytes with concanavalin A, a polyclonal T-cell mitogen, in the presence of GluOC promoted
T cell proliferation and their interferon-γ (IFN-γ) production. Besides, GluOC directly suppressed
B16 cell growth through downregulating phosphorylation levels of RTKs in vitro.
These results indicate that GluOC exerts antitumor effects not only in vitro, but also in vivo via
cellular immunostimulatory effects in B16 mouse melanoma cells.
Key words: GluOC, antitumor effects, immunostimulatory effects, IFN-γ

17

PSS-3
Infection of microglia with Porphyromonas gingivalis promotes cell
migration and an inflammatory response through thegingipain-mediated activation of protease-activated receptor-2
Yicong Liu1, Zhou Wu 1,2, Junjun Ni1, Yoshinori Hayashi1, Fumiko Takayama1,
Yurika Nakanishi1, Yamin Zhou3, Tomoko Kadawaki4, Hiroshi Nakanishi1
1

Department of Aging Science and Pharmacology,
OBT Research Center, Faculty of Dental Sciences, Kyushu University, Fukuoka 812-8582,
Japan, 3Department of Implantology, School of Stomatology, Jilin University, Changchun
130021, China, 4Division of Frontier Life Science, Department of Medical and Dental Sciences,
Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan.
2

Despite a clear cause and effect relationship having been demonstrated between periodontitis andcognitive decline in Alzheimer’s disease, the precise mechanism underlying the relationshipremains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class ofcysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp).
Rgp and Kgp are important in the bacterium-mediated host cell responses and the subsequent
intracellular signaling in infected cells. In the present study, we attempted to crarify the potential
effects of Rgp and Kgp on the cellular activation of brain-resident microglia. We provided the firstevidence that Rgp and Kgp cooperatively contribute to the P. gingivalis-induced cell migration andexpression of proinflammatory mediators through the activation of protease-activated protease 2
and phosphatidylinositol 3-kinase/Akt pathway in microglia.
Key words: Porphyromonas gingivalis, gingipains, microglia, cell migration, neuroinflammation

18

PSS-4
Exploration of IgA-binding bacteria in salivary microbiomeShinya Kageyama, Toru Takeshita, Mikari Asakawa, Yukie Shibata, Rie
Matusmi, Michiko Furuta, Kenji Takeuchi, Yoshihisa YamashitaSection of Preventive and Public Health Dentistry, Faculty of Dental Science, Kyushu University,
Fukuoka 812-8582, Japan
Numerous bacteria inhabit and construct a complex but stable ecosystem in the oral cavity, and are
involved in the progression of dental caries and periodontitis, as well as systemic disorder such as
pneumonia. However, the regulation of their homeostasis remains unclear. In this study, we focusedon immunoglobulin A (IgA) secreted into saliva, and investigated the relationship between IgA andoral microbiome. Stimulated saliva was collected from 8 healthy individuals (26-61 years old).
Their salivary bacteria were stained with APC-labelled anti-human IgA, and sorted into
IgA-negative bacteria and IgA-positive bacteria by cell sorter. The bacterial DNA was extracted
from each pre- and post sorting sample, and their bacterial composition was determined by 16S
rRNA gene sequencing analysis using the next generation sequencer, Ion PGM. Of 342
species-level operational taxonomic unit (OTUs), 22 OTUs corresponding to bacteria such as
Prevotella melaninogenica, Neisseria flavescens, and Streptococcus salivarius exceeded 1% in
pre-sorting samples and constitute 82.0 ± 5.7% of each microbiome. Most of the salivary bacteria(84.3 ± 2.9%) were bound by IgA, whereas 3 OTUs corresponding to Fusobacterium periodonticum,
Lautropia mirabillis, and Veillonella atypica were characteristically uncoated with IgA in saliva of
the most individuals, regardless of the concentration of IgA, age, and sex. These results suggested
that some specific oral bacteria might evade mucosal immunity-recognition.
Key words: salivary microbiome, IgA, 16S rRNA gene, cell sorter

19

PSS-5
Exosomes from oral squamous carcinoma cells define the tropism ofinvasiveness and lymphatic dissemination
Masahiko Morioka , Tomoyo Kawakubo-Yasukochi2, Seiji Nakamura1,
and Manabu Nakashima2
1,2



1

Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan2
Department of Immunological and Molecular Pharmacology, Faculty of Pharmaceutical
Science, Fukuoka University, Fukuoka 814-0180, Japan

Emerging evidence indicates that cancer-derived exosomes increase the tumorigenic potential oftumor cells by reprogramming the cells associated with the tumor microenvironment.
Our study aimed to examine the effect of cancer cell-derived exosomes on invasive andmetastatic process, using two oral squamous carcinoma cell (OSCC) clones, SQUU-A and SQUU-B,
from the same patient. Our data demonstrated that exosomes derived from highly metastatic
SQUU-B cells conferred invasive ability to nonmetastatic SQUU-A cells and subsequently reducedmRNA expression of cytokeratin 13, which is strongly linked to malignant transformation of
OSCCs.
We further examined the effect of the OSCC-derived exosomes on angiogenesis andlymphangiogenesis, closely related with hematogenous and lymphatic metastasis, respectively,
using HUVECs (human umbilical vein endothelial cells) and HDLECs (human dermal lymphatic
endothelial cells). As a result of expression assays, HUVECs exposed to the exosomes had littlechange in the expression levels of VEGFs (vascular endothelial growth factors) and VEGFRs(VEGF receptors), which are closely related with tube formation. In contrast, the expression levelsof VEGFR1, VEGFR2 and VEGFR3 in HDLECs were significantly increased by the exosomes, andthose of VEGF-A, VEGF-C and VEGF-D were increased only by the exosomes from SQUU-B.
Additionally, these results were reflected to structure-forming ability in tube formation assay.
Our data indicate that the cancer cell-derived exosome undertakes crosstalk with different
malignant cell clones in an identical tumor microenvironment and luminal cells closely related tocancer dissemination, and which may define clinical prognosis.
Key words: oral squamous carcinoma, exosome, invasion, matastasis

20

PSS-6The effect of innate micro-inflammation on pancreatic islet cells
Mitsudai Tsuruta, Misaki Iwashita, Shinjo Takanori, Akiko Yamashita, Hiroaki
Matsunaga, Fusanori Nishimura
Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science,
Kyushu University, Fukuoka 812-8582, Japan
BackgroundIn subjects with Type 2 diabetes, infiltration of inflammatory cells such as
macrophages is reportedly observed in islet in addition to the dysfunction of islet cells such as alphaand beta cells associated with disease progression. However, the role of infiltrated inflammatory
cells on islet function remains unclear. We previously reported that the interaction between tissue
infiltrated macrophages and adipocytes or glomerular mesangial cells contributed to thedevelopment of insulin resistance or renal dysfunction through inflammation.
Research objectivesThe aim of this study is to investigate the effect of interaction between
infiltrated inflammatory cells and pancreatic alpha or beta cells on islet function. MethodsMouseadenoma-derived pancreatic α cell line αTC1 or insulinoma-derived pancreatic β cell line βTC6
were co-cultured with mouse macrophage-derived cell line RAW 264.7 using a transwell system.
Changes in gene expression in the co-cultured αTC1 or βTC6 stimulated with E.coli
lipopolysaccharide (LPS) were comprehensively analyzed by DNA microarray method. ThenmRNA and protein expression in these cells were determined by real-time PCR and western blotting.
In addition, LPS-stimulated interferon (IFN) β secretion from macrophages was measured by
ELISA.
ResultsExpression of the genes belonging to Type 1 IFN gene cluster was shown to besignificantly elevated in both co-cultured αTC1 and βTC6 by LPS stimulation. Among these genes,
mRNA expression of X-linked inhibitor of apoptosis protein associated factor 1 (Xaf1), involved in
the induction of apoptosis, was found to be markedly up-regulated particularly in βTC6. Xaf1
protein expression in βTC6 was also significantly elevated by stimulation with IFNβ or addition ofculture supernatant of LPS-stimulated RAW 264.7. Furthermore, RAW 264.7 was found to secrete
IFNβ by LPS stimulation, indicating that Xaf1 expression in the co-cultured βTC6 was induced by
IFNβ secreted from macrophages in response to toll-like receptor (TLR) 4 ligand.
 Conclusions  These results suggest that IFNβ produced from macrophages activated by
micro-inflammation induces Xaf1 expression in pancreatic beta cells, thereby promoting apoptotic
pathway in β cells initially caused by cellular stress such as hyperglycemia and dyslipidemia, which
potentially promotes the progression of diabetes.
Key word: Micro inflammation, Pancreatic beta cell, Xaf1

21

PSS-7
Transgelin mediates the proliferation of human periodontal ligament
cells induced by TGF-β1
Hiromi Mitarai1, Naohisa Wada2, Daigaku Hasegawa3, Shinichiro Yoshida3,
Mai Sonoda1, Atsushi Tomokiyo3, Sayuri Hamano1,4, Suguru Serita,
Hiroyuki Mizumachi, Hidefumi Maeda1,3

1

Department of Endodontology and Operative Dentistry, Faculty of Dental Science, 2Division ofGeneral Dentistry, 3Division of Endodontology, Kyushu University Hospital, 4OBT ResearchCenter, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan

BackgroundHuman periodontal ligament (PDL) cells express transforming growth factor β1(TGF-β1). As TGF-β1 signaling pathway promotes many physiological processes, including cell
growth, differentiation, proliferation, and collagen production in PDL cells, TGF-β1 is thought toplay key roles in homeostasis of PDL tissue. Transgelin, known as a cytoskeleton-associated protein,
has a Smad-binding element in gene promoter, suggesting that transgelin could be a target gene of
TGF-β1 signaling. In this study, we examined the localization and the potential function oftransgelin in PDL tissue and cells.
Materials and MethodsMicroarray analysis of human PDL cell lines (2-14, 2-23, and 2-52) was
performed. Transgelin expression in primary human PDL cells (HPDLCs) was examined byquantitative RT-PCR, immunofluorescence staining and western blotting analysis. The effects of
rhTGF-β1 and TGF-β1 activin receptor-like kinases (ALK5) inhibitor, SB431542, on HPDLCs
were examined by western blotting analysis. The effects of transgelin knockdown by siRNA on the
proliferation of HPDLCs were assessed by WST-1 assay.
Results In microarray and quantitative RT-PCR analyses, transgelin expression level in 2-14
and 2-23 cells, which highly express PDL markers, such as periostin, alkaline phosphatase,α-smooth muscle actin, and type 1 collagen, was significantly higher than that in 2-52 cells.
Immunohistochemical and immunofluorescence staining revealed the expression of transgelin in rat
PDL tissue and HPDLCs. In HPDLCs, rhTGF-β1 upregulated transgelin expression while theinhibition of ALK5 by SB431542 suppressed the TGF-β1-induced transgelin expression.
Furthermore, transgelin siRNA transfection did not promote the proliferation of HPDLCs treatedwith TGF-β1. The expression levels of cyclinA2 and cyclinE1, which regulate DNA synthesis andmitosis through the cell cycle, were also not upregulated in HPDLCs transfected with transgelinsiRNA.
ConclusionTransgelin is expressed in PDL tissue and might be essential for the proliferation ofhuman PDL cells induced by TGF-β1.
Key words: transgelin; TGF-β1; periodontal ligament
22

PSS-8Characterisation of sucrose-independent supragingival plaqueproduced in an in vitro biofilm model

Kota Tsutsumi, Uchiyama A, Ken-ichiro Shibasaki
Section of Oral Health Promotion and Technology, Graduate School of Dental Science,
Kyushu University, Fukuoka 812-8582, JapanA dental plaque is a complex biofilm comprising a consortium of microorganisms. The
accumulation of supragingival plaque causes dental caries and periodontal diseases. The amount ofsugar consumption in Japan has been decreasing, suggesting that the rate of sucrose-independent
plaque formation is increasing in the current Japanese population. We therefore developed an invitro model of a sucrose-independent supragingival plaque to examine the effect of new
anti-cariogenic agents on a sucrose-independent plaque formation. In this study, we characterised asucrose-independent biofilm model in comparison with a 1% sucrose-dependent biofilm model interms of their products' properties physical strength, type of extracellular polymeric substance(EPS) and acidogenic potential.
For testing the physical strength, the biofilm models were treated with two different washingmethods: (i) manual cleaning with different washing frequencies and (ii) mechanical cleaning withdifferent shaking strengths. The type of EPS was evaluated using confocal laser scanningmicroscopy, after fluorescence labelling of either dextran or lectin (concanavalin A: Con A, wheat
germ agglutinin: WGA). The biofilm model was also incubated with a glucose solution ranging
from 0 to 10% for 3 h and then the pH of the solution was evaluated.
The sucrose-independent biofilm model retained more than approximately 70% of its originallevel after ‘performing manual cleaning two times’ and ‘750-rpm mechanical cleaning’, although
the physical strength of the sucrose-independent biofilm model was slightly lower than that of thesucrose-dependent biofilm model. Glucan, an EPS produced by the synthesis from sucrose, wasobserved only in the sucrose-dependent biofilm model, whereas other EPSs were detected by Con A
and WGA in both biofilm models. On the other hand, the decrease in pH was similar level in bothbiofilm models.
These results suggest the sucrose-independent in vitro biofilm model may produce biofilm
without glucan EPSs, but with physical strength and an acidogenic potential at the same level asthat in produced in the sucrose-dependent biofilm model.
Key words: supragingival plaque, physical strength, EPS, acidogenic potential

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