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Program on March 15th, 2007
Welcome SpeechProf. Akifumi AKAMINE, Dean, Faculty of Dental Science, Kyushu University!
!
Special Lecture
Session 1: (Chair: Prof. Kazuaki NONAKA)!
10:00-10:40
1. TGF-beta mediated Msx2 expression controls occipital somites-derived caudal
region of skull development.
Yang CHAI
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern
California!
10:40-11:20
2. Differentially expressed genes detected by cDNA subtraction are implicated in mouse
molar tooth germ development.
Tamotsu KIYOSHIMALaboratory of Oral Pathology and Medicine, Faculty of Dental Science, Kyushu University!
11:20-12:00
3. Molecular Regulation of Tooth Development and Regeneration.
Irma THESLEFF
Institute of Biotechnology, University of Helsinki, Finland!

s!

Session 2: (Chair: Prof. Toshio KUKITA)
13:30-14:10
4. Mesenchymal Stem Cells: Disease and Cure.
Songtao SHICenter for Craniofacial Molecular Biology, School of Dentistry, University of Southern
California
14;10-14:50
5. Molecules implicated in the fate determination of injured neurons.
Hiroshi KIYAMA
Department of Anatomy & Neurobiology, Grad. Sch. of Med., Osaka City University,Japan

14:50-15:00
Break

!

t!

Oral Presentation by Graduate School Students
Session 1: (Chair: H. Maeda,Y. Aida)
15:00-15:45
1. Investigating a clonal human periodontal ligament progenitor/stem cell line invitro and in vivo.
vS. Fujii, H. Maeda, N. Wada, A.Tomokiyo and A. Akamine
Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University
2. Development of a multipotent clonal human periodontal ligament cell line.
vA. Tomokiyo1, H. Maeda1, S. Fujii1, N. Wada2, K. Shima1, and A. Akamine1
1

Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation,

2

Division of General Oral Care, Faculty of Dental Science, Kyushu University

3. Intermittent Force Induces High RANKL Expression in Human Periodontal
Ligament Cells.
v K. Nakao1, T. Goto2*, K.K. Gunjigake1, T. Konoo1, S. Kobayashi2, and K.
Yamaguchi1
1

Division of Orofacial Functions and Orthodontics, and 2Division of Anatomy, Kyushu

Dental College
Session 2: (Chair: T. Fukumoto, I. Kobayashi)
15:45-16:30
4. Possible involvement of cytokines, chemokines, and chemokine receptors in theinitiation and progression of Sjögren’s syndrome.
vM. Moriyama, J. Hayashida, K. Hamanaka, S. Shinozaki, S. Nakamura
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical!

Sciences,

FacultyofDentalu!

Science,

KyushuUniversity

5. Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes
adipocyte differentiation by regulating PPAR! mRNA levels.
vDaigo Yoshiga, Seiji Nakamura, Akihiko Yoshimura
Division of Oral and Maxillofacial Oncology Faculty of Dental Science,
Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation
Kyushu University, Fukuoka, Japan Oral Presentation by Graduate School Students
6. Functional analysis of novel gene, Clone 15 in the tooth morphogenesis.
vK. Fukiwake1,2, T. Kiyoshima1, I. Kobayashi1, M. Xie1, H. Sakai 1
1

Department of Oral Pathology, Faculty of Dental Science, Kyushu University,2

Department of Fixed Prosthodontics, Faculty of Dental Science, Kyushu University

Session 3: (Chair: Y.Ayukawa, M.A. Kido)
16:30-17:15
7. CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interactingwith BMP and Notch signaling pathways.
vT. Minamizato1,2, Kei Sakamoto1, Seiji Nakamura2, A. Yamaguchi1
1

Section of Oral Pathology, Graduate School of Tokyo Medical and Dental University.

2

Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Graduate School of Dental Science, Kyushu University
8. Intra-arterial injected microglia engraft in the brain to rescue neuronal damage
v Y. Hayashi1, J. Yamada1, Z. Wu1, M. Sawada2, and H. Nakanishi1.
1

Laboratory

of

Oral

Aging

Science,FacultyofDentalScience,

KyushuUniversity,2Department of Brain Science, RIEM, Nagoya University
9. Presynaptic Inhibition of Synaptic Inputs Precedes Synaptic Stripping in Vagal
Motoneurons after Axotomy.
vJ. Yamada, Y. Hayashi, and H. NakanishiLaboratory of Oral Aging Science, Faculty of Dental Science, Kyushu University, Japan!

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ABSTRACTS
Special Lecture 1 (10:00-10:40)
TGF-beta mediated Msx2 expression controls occipital somites-derived caudal
region of skull development.
Yang CHAI
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern
California, CA, U.S.A.
Craniofacial development involves cranial neural crest (CNC) and mesoderm-derived
cells.

TGF-b signaling plays a critical role in instructing CNC cells to form thecraniofacial skeleton. However, it is not known how TGF-b signaling regulates the fateof mesoderm-derived cells during craniofacial development. In this study, we show that
occipital somites contribute to the caudal region of mammalian skull development.
Conditional inactivation of Tgfbr2 in mesoderm-derived cells results in defects of thesupraoccipital bone with meningoencephalocele and discontinuity of the neural arch of
the C1 vertebra.

At the cellular level, loss of TGF-b signaling causes decreasedchondrocyte proliferation and premature differentiation of cartilage to bone. Expressionof Msx2, a critical factor in the formation of the dorsoventral axis, is diminished in the
Tgfbr2 mutant. Significantly, overexpression of Msx2 in Myf5-Cre;Tgfbr2flox/flox
mice partially rescues supraoccipital bone development. These results suggest that the
TGF-b/Msx2 signaling cascade is critical for development of the caudal region of theskull.
!
!

!

x!

Special lecture 2 (10:40-11:20)
Differentially expressed genes detected by cDNA subtraction are implicated in mouse
molar tooth germ development.
Tamotsu KIYOSHIMALaboratory of Oral Pathology and Medicine, Faculty of Dental Science, Kyushu University
Mammalian tooth development is mediated by the sequential and reciprocal
epithelial-mesenchymal interactions of various molecules like other organs. In order toscreen the potential genes involved in the initiation of mouse odontogenesis, wepreviously made cDNA subtracted libraries between mouse embryonic day (E) 10.5 andE12.0 mandibles, and found several genes to be differentially expressed in the early
development stage of the mandible. We have already reported that the expressions ofseveral genes among them were associated with the developing tooth germ. In this study,
we picked up three genes, nucleolin, thymosin beta 4 (T"4), and a novel gene, from thelibraries, and possible functions of these genes in the development of tooth germ wereanalyzed. Nucleolin and the novel gene mRNAs highly expressed in the E10.5 mandible,
whereas T"4 mRNA expression was stronger in E12.0 mandible. Both nucleloin andT"4 expressions were seen in the odontogenic epithelial cells in the early stage, and in
the inner enamel epithelial layer in the late stage. An inhibition assay using antisense
phosphorothioated oligonucleotides (AS S-ODN) for each nucleloin and T"4 showed amarked inhibition of odontogenesis in cultured mandibles at E11.0 and/or tooth germs
at E15.0. However, the histological findings after AS S-ODN showed different patterns
depending on the target. A significant reduction in mRNA expressions of some genes
related to them was also observed in the organ cultures after AS S-ODN. These findings
suggest that nucleolin and T"4 play roles in the initiation, growth and differentiation of
tooth germ or odontogenesis-associated genes expression. Similarly a novel gene that
we tentatively called as Clone 15 has been characterized and its expression duringodontogenesis in mouse embryo was examined.

!

y!

Special lecture 3 (11:20-12:00)!
Molecular Regulation of Tooth Development and Regeneration.
Irma THESLEFF
Institute of Biotechnology, University of Helsinki, Finland
The communication between cells and tissues is a major mechanism in the regulation ofembryonic development. In most organs, including the teeth, interactions between theepithelial and mesenchymal tissues are central regulators of morphogenesis. The celland tissue interactions are mediated by secreted signaling molecules belonging to a few
conserved families, and they regulate all aspects of development including growth,epithelial morphogenesis, cell differentiation and deposition of extracellular matrices.
The modulation of signal activities influences the numbers, shape and size of teeth aswell as the formation of the dental hard tissues, and it is believed that the evolution of
the patterns of dentitions and tooth morphologies has largely resulted from thefine-tuning of signal pathways.
The number of teeth and their replacement patterns show remarkable variation invertebrates. In non-mammalian vertebrates, such as fish and reptiles, continuous
renewal of teeth is common. In most mammals tooth replacement has been reduced to
the formation of only two dentitions or reduced altogether as in mice. The continuous
replacement of hairs and feathers has been linked to Wnt signalling. We have shownthat when Wnt signalling is activated in transgenic mice by expressing stabilized
beta-catenin in the oral epithelium, the process of continuous tooth renewal is activated.
Most mammalian teeth do not grow throughout life. However, rodent incisors arecharacterized by the ability to grow continuously throughout the life of the animal.
These teeth have a stem cell niche in their proximal ends, and our recent results indicatethat the growth of mouse incisors can be dramatically altered by modulating theactivities of conserved signal pathways in this niche. We showed that a complex
network involving BMPs, FGFs, Activin and Follistatin regulates the proliferation as
well as differentiation of epithelial stem cells in mouse incisors, and that fine-tuning of
their activities may affect both growth and enamel production in the incisors!

z!

Special lecture 4 (13:30-14:10)
Mesenchymal Stem Cells: Disease and Cure.

Songtao SHICenter for Craniofacial Molecular Biology, School of Dentistry, University of Southern
California, CA, U.S.A.
Mesenchymal stem cells (MSCs) have been identified as a population of organized hierarchicalpostnatal stem cells with the potential to differentiate into multiple cell types. These cells were
initially isolated from bone marrow, named bone marrow mesenchymal stem cells (BMMSCs),
and identified by their capacity to form clonogenic adherent cell clusters with a fibroblastic
morphology (CFU-F: colony-forming units-fibroblast) in vitro. Although BMMSCs are capableof multi-differentiation, it appears that they are most readily encouraged to develop towards anosteogenic pathway. When transplanted into immunocompromised mice subcutaneously,
BMMSCs are capable of forming a bone/marrow organ-like structure. One of the most striking
characteristics of the xenogeneic transplantation system is that BMMSCs can organize andsupport a hematopoietic marrow during the osteogenic process. This may represent a dynamic
process whereby multipotential BMMSCs strive to re-construct the microenvironment fromwhich they were derived. Here we demonstrated how genetic deficient of caspase-3 and beta2
integrin contributes to BMMSC-associated osteoporosis phenotype. Moreover, we show that
BMMSCs possess organizing function to construct a bone/marrow organ-like system (BMOS)
that contains hematopoietic progenitors of recipient origin. These hematopoietic cells expressed
multiple lineages of hematopoietic cell associated markers and were able to rescue lethally
irradiated mice with successful engraftment in the recipient. Importantly, BMMSC-generated
BMOS could be re-transplanted to secondary recipients to provide hematopoietic cells in thecirculation, suggesting a great potential of utilizing this BMOS system for stem cell therapies.
In order to further explore potential of using BMMSCs for clinical therapies, we generated an
mandible osteoradionecrosis (ORN) model in swine (miniature pig) and employed autologous
BMMSC transplantation to cure the ORN, suggesting a novel therapeutic approach to recover
ORN defects by BMMSC-generated new bone and BMMSC-organized new homeostasis
microenvironment. Furthermore, we used autologous transplantation of periodontal ligament
stem cells (PDLSCs), a newly identified orofacial mesenchymal stem cell population, to treat!

{!

periodontitis in swine. PDLSCs are capable of regenerating cementum and Sharpey’s fibers tocure periodontitis. In summary, mesenchymal stem cells may involve in many human diseases
and they are a promising stem cell population for clinical therapies.

!

"|!

Special lecture 5 (14:10-14:50)
Molecules implicated in the fate determination of injured neurons.
Hiroshi KIYAMA
Department of Anatomy & Neurobiology, Grad. Sch. of Med., Osaka City University,
Japan
CNS neurons are susceptible to nerve injury, whereas PNS neurons are relatively
resistant to injury. This fate difference between CNS and PNS could be partly due to thedifferential expressions of molecules in response to nerve injury. Therefore theidentification of molecules in response to nerve injury using PNS injury model isrelevant. In order to identify therapeutic strategies for CNS injury, systematic screeningof the transcriptome and proteome is an effective strategy. We have previously
performed screening assays such as differential display and expressed-sequence-tag(EST) analysis, using the cDNA library from nerve-injured motor nuclei, as well asproteomic analysis. In those studies, we identified several molecules that are induced in
response to peripheral nerve injury. Amongst those molecules I would like to introduce
two molecules whose expression pattern and function in nerve regeneration are
particularly intriguing. One is the Damage-Induced Neuronal Endopeptidase (DINE),
which has been identified as a zinc metalloprotease, and as belonging to the neprilysin
family[1]. Although substrates for this protease are unknown, DINE has unique featuresin its gene expression regulation. The expression of DINE is neuron specific and highly
associated with nerve injury of both CNS and PNS neurons [2]. An analysis of thetranscription mechanism therefore would provide us with a tool for gene therapy.
Another molecule is a glutamate transporter EAAC1 (or EAAT3). Recently we haveidentified that EAAC1 has a unique action in preventing mitochondria-mediatedneuronal death. This “rescue” activity does not depend on glutamate removal, but
requires an interaction with a mitochondrial protein, holocytochrome-c synthetase(HCCS), which is released from the mitochondria and toxic in cytoplasm [3]. This novel
mechanism actually prevents the death of the injured motor neurons of animals, whereas
the failure of the expression of EAAC1 leads to slow degeneration of motor neurons.
The significance of those molecules in the fate determination of injured neurons would!

""!

be discussed.
1. Kiryu-Seo S, et al, Damage induced neuronal endopeptidase (DINE) is a uniquemetallopeptidase expressed in response to neuronal damage and activates superoxide
scavengers. Proc. Natl. Acad. Sci. USA 97(2000) 4345-4350.
2. Kato R, et al, Damage induced neuronal endopeptidase (DINE/ECEL) expression isregulated by LIF and deprivation of NGF in rat sensory ganglia after nerve injury. J
Neurosci 22(2002) 9410-9418
3. Kiryu-Seo S, et al, Unique anti-apoptotic activity of EAAC1 in injured motor neurons,
EMBO J 25(2006) 3411-3421!

"s!

Oral presentation by Graduate School Students (15:00-17-15)
1. Investigating a clonal human periodontal ligament progenitor/stem cell line invitro and in vivo.
vS. Fujii, H. Maeda, N. Wada, A.Tomokiyo and A. Akamine. Kyushu University,
Faculty of Dental Science, Division of Oral Rehabilitation, Fukuoka, Japan
Objectives: The periodontal ligament (PDL) is a highly specialized tissue connecting
the cementum with the tooth socket bone, and affects the life span of tooth. However,
little is known about the precise characteristics and regenerative mechanism of human
PDL, because of the absence of cell lines and specific makers, and the fact that the PDL
tissue consists of heterogeneous cell populations. Therefore, to clarify the regenerative
mechanism of human PDL, we aimed to establish and characterize clonal human PDL
cell lines.
Methods: Normal human PDL fibroblasts (HPLF) were obtained from a healthy firstpremolar of one volunteer. HPLF were transfected with SV40T-Ag gene and hTERT
gene, named as STPLF. The expression of SV40T-Ag and hTERT in STPLF wasascertained by RT-PCR analysis and Western blotting analysis. Twenty clonal human
PDL cell lines were obtained from a limiting dilution of STPLF. Three of them, lines
1-4, 1-11, and 1-24, which were used in this study, were examined on the expression of
STRO-1 and CD146 immunocytochemically, and the gene expression by RT-PCR
technique. Moreover, line 1-11 was examined on the potential to differentiate into
osteoblastic or adipocytic cells in vitro, and to construct PDL-like tissues in SCID mice.
Results: STPLF obviously expressed SV40T-Ag and hTERT, and showed stableproliferation at more than 120 population doublings (PD), while HPLF stopped at 20
PD. Lines1-4, 1-11, and 1-24 expressed RUNX2, Col I, ALP, OPN, OCN, RANKL, OPG,
Scx, Periostin, Col XII, and !-SMA mRNA, comparable to HPLF. Immunocytochemical
data showed that CD146 was expressed in lines 1-4 and 1-11, and STRO-1 was
expressed in lines 1-11 and 1-24. Lines 1-4 and 1-11 cultured in lineage-specificdifferentiation media differentiated into osteoblastic cells and adipocytic cell. When line
1-11 was transplanted into SCID mice with "-TCP for eight weeks, the transplant
formed PDL-like tissues on the surface of !-TCP.
!

"t!

Conclusion: These results suggest that the clonal human PDL cell line 1-11 wasderived from a progenitor/stem cell present in PDL. This clone should be a very helpful
tool for studying the regeneration mechanism of human PDL and for developing new
therapies for periodontitis.

!

"u!

2. Development of a multipotent clonal human periodontal ligament cell line.
vAtsushi Tomokiyo1, Hidefumi Maeda1, Shinsuke Fujii1, Naohisa Wada2, Kazuya
Shima1, and Akifumi Akamine1
1

Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation,

2

Division of General Oral Care, Faculty of Dental Science, Kyushu University

Objectives: The development of new PDL-regenerative therapies requires the isolationof PDL stem cells. However, their characteristics are unclear because of the fact thatPDL is composed of complex cell populations and the absence of somatic PDL stem
cell lines. Therefore, we aimed to establish a human somatic PDL stem cell line andinvestigate the effects of bFGF on its osteoblastic differentiation.
Methods: A clonal human PDL cell line, designated as line 1-17, was exposed toosteoblastic, neurocytic, chondrocytic, and adipocytic induction. To investigate theregulatory mechanism of osteoblastic differentiation of line 1-17 by bFGF, it wascultured in osteoblastic differentiation medium with bFGF in various treatment lengths.
Results: Line 1-17 differentiated into osteoblastic, neurocytic, chondrocytic, andadipocytic cells, which expressed embryonic stem cell (ESC)-related pluripotency genes
OCT3/4 and Nanog, as well as PDL-related moleclules periostin and scleraxis.
Continual bFGF treatment of line 1-17 in osteoblastic induction medium inhibited its
calcification, whereas the delayed addition of bFGF potentiated its calcification and
ALP and OCN mRNA expression. Furthermore, the addition of bFGF to a co-culture ofline 1-17 with Saos2 induced calcification in line 1-17.
Discussion: Our present results suggest that line 1-17 is a PDL-committed stem cell line,that bFGF exerts biphasic (i.e., promotive and inhibitory) effects on osteoblastic
differentiation of line 1-17 according to its differentiation stage, and that bFGF acts on
osteoblasts to induce osteogenesis of line 1-17, possibly via osteo-inductive factors
secreted by them.

!

"w!

3. Intermittent Force Induces High RANKL Expression in Human Periodontal
Ligament Cells.
v K. Nakao1, T. Goto2*, K.K. Gunjigake1, T. Konoo1, S. Kobayashi2, and K.
Yamaguchi1
1

Division of Orofacial Functions and Orthodontics, and 2Division of Anatomy, Kyushu

Dental College
In clinical orthodontics, an orthodontic force is routinely applied for months. Boneremodeling occurs during orthodontic tooth movement, including both bone resorption
on the compression side and bone formation on the tensile side. Regulation of boneregeneration during orthodontic treatment is crucial for the success of orthodontic
treatment. While previous studies have focused mainly on the expression ofbone-related genes, such as RANKL or RUNX2, little attention has been paid to the cell
damage caused by mechanical stress. This study examined the molecular mechanism of
the remodeling of human periodontal ligament (PDL) cells stimulated by an intermittent
force that exceeded the expected value predicted by the duration of force applicationcompared with a continuous force.
Human PDL cells were isolated from premolars extracted for orthodontic reasons.
The PDL cells were subjected to a compressive force (2.0 or 5.0 g/cm2) for 2-4 days by
increasing the amount of medium. A continuous force was applied all day or anintermittent force for 8 h per day. Total RNA was extracted from PDL cells after 2, 3,
and 4 days, and we examined RANKL, OPG, and IL-1" mRNA expression usingsemi-quantitative RT-PCR. Cell damage was assessed by measuring the activity oflactic dehydrogenase (LDH) released from the cells.
At days 3 and 4, there was less cell damage with the intermittent force than with thecontinuous

force.

Compressed

PDL

cells

expressed less OPG mRNA, in a force- andtime-dependent manner. At day 4, the expressionof RANKL and IL-1" was greater with theintermittent force. An IL-1 receptor antagonist
inhibited!

thecompressive

force-induced"x!

RANKL expression.
Based on these findings, we propose the following mechanism to explain how acompressive force induces osteoclastogenesis in PDL cells. An intermittent force
promotes IL-1" expression; the released IL-1" binds to the IL-1" receptor, and thissignal enhances RANKL expression, inducing osteoclastogenesis. Consequently, a
weak intermittent force effectively induces expression of RANKL via IL-1" expression
in human PDL cells, resulting in less overall damage.

!

"y!

4. Possible involvement of cytokines, chemokines, and chemokine receptors in theinitiation and progression of Sjögren’s syndrome.
vMasafumi Moriyama, Jun-nosuke Hayashida, Keiko Hamanaka, Shouichi Shinozaki

Seiji Nakamura
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University

Objective: Sjögren’s syndrome (SS) is an autoimmune disease characterized bylymphocytic infiltration into the salivary and lacrimal glands with concomitant
destruction of the glandular tissue. Although the etiology and pathogenesis of SS is not
fully understood, an involvement of cytokines, chemokines, and chemokine receptors
in the pathogenesis of SS has been suggested. In this study, we thus addressed toclarify the involvement of that in the initiation and progression of SS
Methods: Expression of these molecules in the labial salivary glands (LSG) andperipheral blood mononuclear cells (PBMC) from 47 SS patients and 13 healthy
volunteers was examined by real-time polymerase chain reaction-based method andimmunohistochemical analysis.
Results: The mRNA expression of T helper 1 (Th1) and Th2 cytokines, and several
chemokines and chemokine receptors in the LSG from SS patients was significantly
increased in comparison with that in the PBMC from SS patients, and also with that in
the LSG and PBMC from the healthy volunteers. The mRNA expression of Th2
cytokines, macrophage-derived chemokine (MDC), thymus-and activation-regulated
chemokine (TARC), and CC chemokine receptor 4 (CCR4) in the LSG from SS
patients was closely associated with a strong T cell infiltration in the LSG. Furthermore,
MDC and TARC were immunohistochemically detected in or around ductal epithelial
cells in the LSG, while CCR4 was on the infiltrating lymphocytes in the LSG.
Conclusions: These results suggest that SS might be initiated and/or maintained by!

"z!

Th1/Th0 cells and thereafter progresses in association with Th2 cells accumulation via
the interaction of particular chemokines and chemokine receptors.

!

"{!

5. Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes
adipocyte differentiation by regulating PPAR! mRNA levels.
!
vDaigo Yoshiga, Seiji Nakamura, Akihiko Yoshimura
Division of Oral and Maxillofacial Oncology, Faculty of Dental Science, Kyushu
University
Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation,
Kyushu University

Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by
activating their receptor tyrosine kinase.

SH2-B is an adaptor protein containing

pleckstrin homology (PH) and Src homology 2 (SH2) domains that have been
implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link
between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic
genes including peroxisome proliferator-activated receptor g (PPARg) were reduced inwhite adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of
SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response toinsulin and dexamethasone, while retroviral SH2-B overexpression enhanced
differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced
mRNA level of PPARg in 3T3-L1 cells, while PPARg levels were reduced in SH2-B
deficient MEFs in response to insulin. SH2-B-mediated upregulation of PPARg mRNAwas blocked by a PI3-kinase inhibitor, but not by a MEK inhibitor. Insulin-induced Akt
activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a
negative regulator of PPARg transcription, were upregulated by SH2-B overexpression,
but reduced in SH2-B-deficeint MEFs. These data indicate that SH2-B is a key
regulator of adipogenesis both in vivo and in vitro by regulating the insulin / IGF-I receptor - Akt - Foxo1 - PPARg pathway.

!

s|!

6. Functional analysis of novel gene, Clone 15 in the tooth morphogenesis.
vKeiko Fukiwake1,2, Tamotsu Kiyoshima1, Ieyoshi Kobayashi1, Ming Xie1,
Hidetaka Sakai 1,
1

Laboratory of Oral Pathology and Medicine, Faculty of Dental Science, Kyushu

University
2

Department of Fixed Prosthodontics, Faculty of Dental Science, Kyushu University

Objective: We previously performed cDNA subtraction between the embryonic day
10.5 (E10.5) and E12.0 mouse mandibles to detect factors which are related to the earlystage of tooth morphogenesis, and found differentially expressed genes in eachmandible. The expressions of some genes were apparently associated with thedeveloping tooth germ. Clone 15 was a novel gene of them. The function of this gene
was not yet been elucidated. The aim of this study is to identify and clarify thefunctional implication of this gene in the tooth morphogenesis.
Experimental procedures & Results: At first, we confirmed the sequence of this geneand performed structure analysis according with the deduced amino acid sequence.
Clone15 gene belongs to the immunoglobulin superfamily and showed significant
homology to DEAL (DCC et al).
Next, we generated anti-Cl.15 polyclonal antibody, and tried to prove an existence of
this protein. Immunoblotting revealed this protein to be seen in E10.5 mouse mandible,and highly glycosylated. Furthermore, we examined expression pattern of Cl.15 protein
by immunohistochemistry, and compare with that of Clone.15 mRNA by in situ
hybridization in developing mouse tooth germ (first molar tooth). As a result, strong
signals of both protein and mRNA were expressed at early stage of embryonic days,
especially at E10.5. The expression of Clone15 protein and Cl.15 mRNA in tooth germwas getting weakly day by day. While, in other organs, signals were seen in nervous
systems during embryonic days, these expression patterns were very similar to DEAL.
Conclusion: Clone 15 actually existed as a functional gene that encoded Clone 15
protein, and appeared at early stage of tooth morphogenesis. Therefore, Clone15 may
play important roles to guide a thickening of epidermis as a start point of toothmorphogenesis. Because Clone 15 was also observed at nervous systems, it was!

s"!

suggested that Clone 15 has some relation to functions of DEAL.

!

ss!

7. CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interactingwith BMP and Notch signaling pathways.
vT. Minamizato1,2, Kei Sakamoto1, Seiji Nakamura2, A. Yamaguchi1
1

Section of Oral Pathology, Graduate School of Tokyo Medical and Dental University.

2

Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Graduate School of Dental Science, Kyushu University

We elucidate the role of NOV/CCN3, a member of the CCN family proteins, inosteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3
adenovirus (AdCCN3) alone induced no apparent changes in the expression of
osteoblast-related

markers,

whereascotransduction

with

BMP-2

adenovirus(AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA
expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western
analysis revealed that CCN3 associated with BMP-2. Compared to transduction withAdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expressionof phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with
AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1
and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effectsof CCN3 on the expression of BMP-2-induced osteoblast-related markers were
nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts
inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of
the BMP and Notch signaling pathways.

!

st!

8. Intra-arterial injected microglia engraft in the brain to rescue neuronal damage.
v Yoshinori Hayashi1, Jun Yamada1, Zhou Wu1, Makoto Sawada2, and Hiroshi
Nakanishi1
1

Laboratory of Oral Aging Science, Faculty of Dental Science, Kyushu University,

Japan
2

Department of Brain Science, RIEM, Nagoya University, Japan

There is accumulating evidence that transplanted bone marrow cells recruit to the brain
parenchyma as microglia to ameriolate neuronal damage. In the present study, we havethus attempted to elucidate the effects of the intra-arterial injection of microglia on the
global ischemia-induced functional and morphological deficits of hippocampal CA1
neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected
into the subclavian artery, these exogenous microglia were found to accumulate in the
hippocampus at 24 h after ischemia. In hippocampal slices prepared frommedium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including
a significant reduction of synaptic responses and a marked reduction of long-term
potentiation (LTP) of the CA3–CA1 Schaffer collateral synapses were observed. At thisstage, however, neither significant neuronal degeneration nor gliosis was observed in
the hippocampus. At 96 h after ischemia, there was a total loss of the synaptic activity
and a marked neuronal death in the CA1 subfield. In contrast, the basal synaptictransmission and LTP of the CA3–CA1 synapses were well preserved after ischemia inthe slices prepared from the microglia-injected animals. We also found themicroglial-conditioned medium (MCM) to significantly increase the frequency of the
spontaneous postsynaptic currents of CA1 neurons without affecting the amplitude, thus
indicating that MCM increased the provability of the neurotransmitter release. The
protective effect of the intra-arterial injected microglia against the ischemia-induced
neuronal degeneration in the hippocampus was substantiated by immunohistochemical
and immunoblot analyses. Furthermore, the arterial-injected microglia prevented theischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in
CA1 neurons. These observations strongly suggest that the intra-arterial injected
microglia engraft in the brain to rescue neuronal damage.
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9. Presynaptic Inhibition of Synaptic Inputs Precedes Synaptic Stripping in Vagal
Motoneurons after Axotomy.
vJun Yamada, Yoshinori Hayashi, and Hiroshi NakanishiLaboratory of Oral Aging Science, Faculty of Dental Science, Kyushu University, Japan
Microglia are representatives of the resident mononuclear phagocyte population within
the central nervous system. They secrete soluble factors including neurotrophins to
support neuronal survival and functions. There is accumulating evidence that activatedmicroglia also protect neurons by physically removing synaptic inputs. Following an
axotomy, microglia are rapidly activated and then transform into a deramified form, andthey thereafter proliferate, adhere to regenerating motoneurons, and spread on theirsurface. This phenomenon is now known as “synapic stripping”. It is believed that
activated microglia spread on the motoneurons engage in the displacement of detachedafferent synaptic boutons from the surface of a regenerating neuron. It is widelybelieved that synaptic stripping plays an important role in neuronal survival and axonal
regeneration of injured motoneurons. However, little information is available for thedetailed pathological significance of synaptic stripping. In the present study, we havefirst attempted to examine whether changes in synaptic inputs after an axotomy arecorrelated with synaptic stripping in the dorsal motoneurons of vagas (DMV) of rats. At
2 days after an axotomy, there was a significant decrease in frequencies of bothspontaneous EPSCs and IPSCs recorded from DMV without significant changes in theiramplitudes. However, synaptic stripping was not evident at this stage. Furthermore,high-K+ condition restored frequencies of both spontaneous EPSCs and IPSCs to thecontrol levels. These results strongly suggest that presynaptic inhibition of synaptic
inputs precedes synaptic stripping in DMV after an axotomy. Therefore, it is reasonable
to consider that some soluble factors inhibit release of glutamate and/or GABA by thepresynaptic mechanism. ATP was found to be one of potential candidates for such
soluble factors because PPADS, a P2 receptor antagonist, recovered the frequency of
spontaneous EPSCs recorded from the axotomized DMV to the control level.
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