Basic Research—Biology

Gene Profiles during Root Canal Treatment inExperimental Rat Periapical Lesions
Zulema Rosalia Arias Martinez, DDS,* Koji Naruishi, DDS, PhD,*
Keisuke Yamashiro, DDS, PhD,* Fumio Myokai, DDS, PhD,* Teruo Yamada, PhD,†
Kaori Matsuura, DDS, PhD,* Naoko Namba, DDS,* Hideo Arai, DDS, PhD,*
Junzo Sasaki, MD, PhD,† Yoshimitsu Abiko, DDS, PhD,‡ and Shogo Takashiba, DDS, PhD*
Abstract
The purpose of this study was to profile gene expression in periapical lesions during root canal treatment(RCT). Periapical lesions were induced experimentallyby exposing the pulp in Sprague-Dawley rats. After 3
wk, the animals received root canal filling (RCF) andwere sacrificed 1 or 4 wk later. From the periapicaltissues, total RNA was extracted and processed for
cDNA-microarray analysis. The lesions were histologically and radiographically confirmed to expand 4 wk
after pulp exposure (inflammation phase) and to stabilize 4 wk after RCF (healing phase). In approximately
30,000 genes on the microarray, 203 genes were upregulated to more than 5-fold (e.g., IL-1␤), and 864
genes were down-regulated to less than 20% of baseline level (e.g., caspase 8) in inflammation phase. Compared with inflammation phase, we found that 133
genes were up-regulated (e.g., IL-1␣) and 50 genes
were down-regulated (e.g., defensin ␣5) in healing
phase. Corresponding to the gene expression profiles,
accumulation of IL-1␣ and IL-1␤ was observed in theperiapical lesions by immunohistochemistry. These
gene profiles might be useful in diagnosing the healing
process of periapical lesions. (J Endod 2007;33:
936 –943)

Key Words
Gene profiles, IL-1␣, IL-1␤, periapical lesion, root canaltreatment

From the *Department of Pathophysiology–Periodontal
Science, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama, Japan; †Department of Cytology and Histology, Okayama University
Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Okayama, Japan; ‡Department of Biochemistry andMolecular Biology, Nihon University School of Dentistry at
Matsudo, Chiba, Japan.
Address requests for reprints to Dr. Shogo Takashiba, DDS,
PhD, Department of Pathophysiology–Periodontal Science,
Okayama University Graduate, School of Medicine, Dentistry
and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama
700-8525, Okayama, Japan. E-mail address: stakashi@cc.
okayama-u.ac.jp.
0099-2399/$0 - see front matter
Copyright © 2007 by the American Association ofEndodontists.
doi:10.1016/j.joen.2007.04.016

936

Arias Martinez et al.

Periapical periodontitis is mainly caused by bacterial infection within the root canal(1), and periapical tissue destruction is caused by endogenous inflammatory mediators released during interaction between bacteria and host cells, mainly polymorphonuclear neutrophils (PMNs), lymphocytes, plasma cells, and macrophages (2).
However, the pathological mechanisms that are responsible for apical tissue destruction such as bone degradation are not fully understood.
After developing periapical lesions, endodontic treatment (root canal treatment,
RCT) has been performed traditionally to remove the infectious agents in root canal (3).
By means of RCT, inflammatory reactions against infectious antigen in periapical lesions
decrease gradually, and lead to the healing process (2, 3).
Clinically, judgment of the success of RCT is abundant but inconclusive. In many
clinical cases, the success has been evaluated by radiographic healing of periapicallesions (4). Also, it may be evaluated by a decrease in clinical symptoms such as
spontaneous pain, percussive pain, and pus discharge from canal. Considering the
inflammatory responses in periapical lesions, success should be evaluated by decrease
in inflammatory reactions. However, molecular mechanisms regulating inflammatory
processes, especially the healing process after RCT in periapical lesions, are still poorly
understood.
Comprehensive gene expression analysis with cDNA microarray is a powerful tool
to investigate the etiology and pathophysiology of diseases including periapical lesions(5, 6). The altered gene expression pattern detected by cDNA microarray analysis
suggests that changes of complex inflammatory reactions during RCT are likely to be
involved in the development and consequences of the disease. In the present study, weestablished an in vivo model of the periapical lesions with RCT to clarify the molecularpathophysiology of the disease. Using cDNA microarray analysis, we report for the firsttime a global gene expression analysis in periapical lesions with RCT. We analyzed thegene expression profiles in periapical lesions to clarify the molecular mechanisms ofthe disease, and we also investigated changes in gene expression relating to the therapeutic effects. The possible targets for diagnosis of healing process in periapical lesions
using immunohistochemical analysis were determined from these data. Our approachshould contribute to the development of novel diagnostic systems in periapical lesions
during RCT.

Materials and Methods
Animals
Male Sprague-Dawley (SD) rats 10 weeks of age were purchased from CLEA Japan(Tokyo, Japan). Rats were fed standard rodent chow and water ad libitum, and housed
under a 12-hour light– dark cycle. Rats were acclimated to the animal facility for 5 days
before any experimental handling. The protocol was approved by the Animal Care
Committee of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.
Experimental Animal Models of Periapical Lesions During RCT
The animals were anesthetized with sodium pentobarbital (50 mg/kg; nembutal
sodium: Dainippon-Sumitomo Pharma, Osaka, Japan). Periapical lesions were induced

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Basic Research—Biology
experimentally by exposing the pulp in the lower first molars of the rats.
After 3 wk, root canals were mechanically disinfected with reamers andfiles, and irrigated with standard saline solution, then root canals were
obturated using gutta-percha point (Showa Yakuhin Kako, Tokyo, Japan) and sealer (Canals N, Showa Yakuhin Kako) with lateral condensation technique. Finally the occlusal cavity was sealed with glass-ionomer cement (Fuji Ionomer Type II: GC, Tokyo, Japan). According toexperimental protocol (Fig. 1A), 4 rats were assigned to 3 groups: anontreatment group (healthy, n ⫽ 4; 3 for RNA sample, 1 for tissue
section); an inflammation group (inflammation, n ⫽ 4; 3 for RNA
sample, 1 for tissue section); and an RCT group (healing, n ⫽ 4; 3 for
RNA sample, 1 for tissue section). Inflammation group has not received
RCT throughout experiments. Periapical inflammatory reactions were
evaluated by radiographic examination in a time dependent mannerusing a portable X-ray machine (MAX-DC 70: Morita, Osaka, Japan). At
the same time, periapical tissues were evaluated histologically in eachgroup of rats.

RNA Preparation
In the three specimens from each group, tissues surrounding periapical area were dissected out with the use of a low-speed handpiecebur and excavator. The samples were immediately inserted in RNAlater(Ambion, Austin, TX) to perform microarray analysis. Then the samplesof each group were pooled into 1 tube and homogenized using a Mixer

Figure 1. Establishment of in vivo experimental model of periapical lesions with
RCT. (A) Experimental protocol. (B) Representative radiographical (X-ray, top
panel) and histological (hematoxylin and eosin staining, middle and bottom
panel) evaluation in each group. In radiographic data in top panel, arrow heads
show the radiolucent area. We stained 3 sections on a glass slide for each group,and selected the most representative view. In the inflammation phase, infiltrated
leukocytes were observed. Scale bars: middle panel, 500 ␮m; bottom panel,
100 ␮m.

JOE — Volume 33, Number 8, August 2007

Mill MM 301 (Retsch, Haan, Germany) and total RNA was isolated using
RNeasy Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany). The RNA
integrity was determined using Agilent 2100 bioanalyzer, and RNA6000
Pico LabChip Kit (Agilent, Palo Alto, CA) and the ratio of the amount of
28S to 18S ribosomal RNAs approached about 2:1.

Microarray Analysis
Rat genome 230 2.0 arrays, which contained approximately
30,000 probes corresponding to known rat genes, were purchasedfrom Affymetrix (Santa Clara, CA). For cDNA microarray analysis, aseries of reverse-transcription, second-strand cDNA synthesis, andprobe generation were accomplished and processed according to manufacturer’s instruction (Affymetrix). Briefly, first-strand cDNA was synthesized with SuperScript II and a T7-(dT)24 primer from total RNA andthen double-strand cDNA was synthesized with Escherichia coli RNase
H, E. coli DNA polymerase I, and E. coli DNA ligase (all from Affymetrix). After the double-stranded cDNA were purified with GeneChip
Sample Cleanup Module (Affymetrix), cRNA was prepared using a
MEGAscript T7 kit (Ambion). After a second cycle of amplification andbiotin labeling with a GeneChip IVT labeling kit (Affymetrix), 20 ␮g oflabeled cRNA was fragmented by mild alkaline treatment, at 94°C for 35
min with GeneChip Sample Cleanup Module (Affymetrix). The Rat genome 230 2.0 arrays were hybridized as described in the GeneChip
Expression Analysis Technical Manual (Affymetrix). After hybridization,
the microarray was rinsed with 0.2 ⫻ SSC (3 mmol/L NaCl, 0.3 mmol/L
Na-citrate) and then scanned with an array scanner (Scanner 3000,
Affymetrix). Scanned images were analyzed using GeneChip Operating
Software (Version 1.1: Affymetrix) and GeneSpring software (version
7.2: Tomy Digital Biology, Tokyo, Japan). In brief, the hybridization
efficiencies were equalized by median value of control probe set to eacharray, and the genes with higher signal intensities than bioB derived
from E. coli as an internal control for hybridization were subjected forfurther analysis. The genes (probe sets) showing greater than a foldchange greater than 5.0 or less than 0.2 were selected as changed genes.
Next, pathway analysis was performed using Ingenuity pathways analysis(IPA) software (www.ingenuity.com, Ingenuity® Systems, Inc, CA), a
web-delivered application that enables biologists to discover, visualize,
and explore potentially relevant pathobiologic networks. Data sets containing the genes accession numbers and their corresponding expression fold-change values were imported into IPA software (Ingenuity
Systems, Inc.). Focus genes identified by the IPA program were categorized based on location, cellular components, and reported biochemical, biologic, and molecular functions using the software. The program
queried the Ingenuity Systems’ Pathway Knowledge Base for interactions
between the focus genes and all other gene objects stored in the knowledge base, and generated a set of networks ranked by score. The scoreis the negative log of a p value and indicates the likelihood of the focus
genes being found in the networks caused by random chance. A score of
3 indicates that there is a 1/1,000 chance that the focus genes are in the
network due solely to chance. Therefore, scores of 3 or greater represent a 99.9% confidence level. Biological functions were then calculated
and assigned to each network.
Quantitative RT-PCR
The quantitative reverse transcription polymerase chain reaction(qRT-PCR) was performed using Chromo 4 Real-time PCR Detector(Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.
Briefly, 5 ␮g of total RNA extracted from the periapical samples werereverse-transcribed with oligo (dT)12–18 primer, dNTPs and Superscript II (all from Invitrogen, Carlsbad, CA) in a total volume of 20 ␮l,
and 0.1 ␮l of this reaction mixture was mixed with both SYBR GreenPCR Master Mix (Applied Biosystems, Foster City, CA) and the geneGene Profiles during Root Canal Treatment937

Basic Research—Biology
specific primers. The PCR primers were designed by Primer 3 (http://
frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and have beenshown in a supplementary Table. The real-time PCR was performed
using Chromo 4 Real-time PCR Detector (Bio-Rad) according to the
manufacture’s instructions. The all amplification of selected genes conditions consisted of an initial 10 min denaturation step at 95°C followed
by 40 cycles of denaturation at 95°C for 15 sec, annealing and elongation at 55°C for 1 min. Three independent assays were performed foreach gene, and we deleted outlier from the further analysis. ␤-Actin wasused as an internal control for the relative quantification of the target
messages, because there were no significant difference in ␤-actin mRNAlevel among the samples by preliminary experiments using the microarray and real-time PCR.

Immunohistochemistry
To confirm the Microarray data, one rat in each group was sacrificed, and periapical area with tooth of each rat was harvested carefully
for histological examination. Tissues were fixed in 4% paraformaldehyde and decalcified by K-CX decalcifying solution (FALMA, Tokyo,
Japan). The decalcified tissues were embedded in paraffin wax, andthree sagittal sections 5-␮m thick were cut and placed together on one
glass slide for histological (hematoxylin and eosin staining) and immunohistochemical examination.
The tissue sections were deparaffinized in xylene and rehydrated.
Sections were rinsed in phosphate-buffered saline solution (PBS) (⫺)
and incubated with 3% H2O2 in methanol for 20 min to quench endogenous peroxidase activity and the sections were rinsed with PBS (⫺).
Primary antibodies were incubated 30 min at room temperature in ahumid chamber. Primary antibodies used in this study were rat anti–
IL-1␣ antibody (Cat. No. sc-9983, Santa Cruz Biotechnology, Santa
Cruz, CA) and rat anti–IL-1␤ antibody (Cat. No. sc-7884, Santa Cruz).
After sections were rinsed with PBS containing 0.2% Tween 20 (PBST),
peroxidase-conjugated goat anti-rat immunoglobulin (Nichirei, Tokyo,
Japan) was incubated for 30 min at room temperature and rinsed with
PBST. Finally, the sections were developed with 3, 3=-diaminobenzidine
tetrahydrochloride (DAB: Vector Laboratories, Burlingame, CA) andmounted with coverslips.
Statistical Analysis
Statistical analysis in quantitative RT-PCR (qRT-PCR) were carriedout using StatView 5.0 software (Abacus Concepts, Berkeley, CA). Comparisons between two groups were performed with unpaired Student’s
t test, and p values of less than 0.05 were considered statistically significant.

Results
Establishment of Experimental Animal Models of
Periapical Lesions
Experimental protocol has shown in Fig. 1A. To confirm the establishment of inflammatory reaction in periapical area, we examinedthe degree of the inflammation by both radiographic and histologic
examination (Fig. 1B). We observed that there was a dramatic inflammatory reaction, infiltration of leukocytes, and thickness of periodontal
ligament 4 wk after pulp exposure in periapical area. Further, the inflammatory reaction decreased slightly 4 wk after RCF as expected.
From these results, we categorized the inflammatory stages around periapical region to three groups (Healthy, Inflammation, and Healing). No
clinical signs of infection or mortality were observed throughout experiments. There were no significant differences in body weight of rats
among each group.
938

Arias Martinez et al.

Microarray Analysis
Global gene expression patterns were generated from Affymetrix
Genechip microarrays using total RNA isolated from periapical tissuesof each group. We compared the gene expression patterns among eachgroup (Healthy vs. Inflammation, Healthy vs. Healing, and Inflammation
vs. Healing) and used a 5-fold change in expression as the experimental
cut-off value that would imply significance. In the 30,000 genes on thearray 203 genes were up-regulated to more than 5-fold (IL-1␤, MMP12, PDGF-␣, etc.) and 864 genes were down-regulated to less than 20%
of baseline level (Cathepsin E, Caspase 8, Calmodulin 3, etc.) in the
inflammation compared with the healthy control (Table 1). In addition,
410 genes were up-regulated to over 5-fold (IL-1␤, Cathepsin L, Integrin ␤6, etc.) and 826 genes were down-regulated to less than 20% of
baseline level (MMP-8, Defensin Rat NP-3 precursor, Cyclin E, etc.) in
the healing phase compared with the healthy control (Table 2). Furthermore, 133 genes were up-regulated to over 5-fold (IL-1␣, MMP-3,
Integrin ␤6, etc.) and 50 genes were down-regulated to less than 20%
of baseline level (Mast cell protease 9, Defensin Rat NP-3 precursor,
Defensin-␣5, etc.) in the healing phase compared with the inflammation (Table 3). To validate the alternation of gene expression levels thatwere detected on the microarrays, qRT-PCR analysis was performed
using RNA obtained from the same tissue samples used in the microarray experiment (Fig. 2). Because the amount of tissue was limited, RNAof the rats belonging to the same experimental group was pooled. As
shown in Fig. 2, we analyzed several genes, IL-1␤, caspase 8, cathepsin
L, IL-1␣, and defensin-␣5 using qRT-PCR. Besides caspase 8 gene, we
demonstrated that gene expression patterns correlated, but generally
higher fold-changes were observed in qRT-PCR experiments comparedwith those obtained in the microarray experiments.
The ingenuity analysis shows possible interactions among the geneproducts which were up- or down-regulated in the microarray experiments. As seen in supplementary Fig. 1–3, the pathway analysis revealed
that interactions of genes during RCT are complex as expected. Especially, we found that increase of IL-1␣ gene is an important factor inhealing phase after the inflammation phase (Supplementary Fig. 3).
Also, we found that the tumor necrosis factor (TNF) gene and transforming growth factor (TGF) ␤1 gene might play a central role in healing phase, although the expression of their genes does not show obvious
change during RCT.
Expression of IL-1␣ and IL-1␤
The pathways presented in the supplemental figures are possible
interactions but do not directly implicate IL-1␣ and IL-1␤ in the processof healing. Since both IL-1␣ and IL-1␤ are known to be involved in theprocess of inflammation (7), we investigated further their expression at
protein levels. The expression of IL-1␣ or IL-1␤ protein in rat periapical lesions was assessed by immunohistochemistry (IHC). As shown in
Fig. 3, the strong staining of IL-1␣ was observed in periapical lesions at
healing phase compared with inflammation phase. Furthermore, we
observed that there was no significant difference between inflammation
phase and healthy controls in IL-1␣ staining. On the other hand, thestrong staining of IL-1␤ was observed in periapical areas at inflammation phase compared with healthy controls. Moreover, we observed that
there was no significant difference between inflammation phase andhealing phase in IL-1␤ staining.

Discussion
Inflammatory processes are thought to play an important role in
the pathogenesis of periapical periodontitis (7). An accumulation ofvarious cell types, neutrophils, macrophages, and lymphocytes in theperiapical lesions has been observed to be associated with the develop-

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Basic Research—Biology
TABLE 1. Gene Profiles in Inflamed Tissue Compared with Healthy Control
Function
Up-regulated (total 203 genes)
Cytokine-related
Enzymes

Growth factor–related

Kinases
Cellular matrix

Transcription regulator
OthersDown-regulated (total 864 genes)
Cytokine-related
Enzymes

Growth factor–related
Kinases

Cellular matrix

Transcription regulator
OthersGene Name

Accession #Fold Change

Chemokine (CXC motif) ligand 1*IL-1␤
Colony stimulating factor 1
MMP-12
Plasminogen activator
Prostaglandin-endoperoxide synthase 2
MMP14
Carboxypeptidase X1
Lysyl oxidase
Insulin-like growth factor binding protein-5
PDGF-␣
Insulin-like growth factor-2
Epidermal growth factor
Fibroblast growth factor-3
MAPKKKK
CaM-kinase II inhibitor ␣
Collagen type XVIII ␣1
Laminin ␣1
Collagen type XIV ␣1
Procollagen, type XV
Cadherin 13
Runt related transcription factor 2
Jun-B oncogene
Heat shock 70kD protein 1A
Fibulin 2
Myosin heavy chain 11
Dynamin 1

NM_030845
NM_031512
AW535553
NM_053963
NM_013151
U03389
AI176440
AI577849
NM_017061
BF399783
AA866419
BI299621
NM_022294
BF410980
BE117850
AI236027
AI101782
BI281952
AI599143
AA800298
NM_138889
BF403180
NM_021836
NM_031971
AA944398
X16262
NM_080689

170.00
6.48
5.22
54.2
15.7
8.99
8.31
7.72
6.43
27.6
10.5
6.56
6.18
5.06
9.65
6.94
9.79
9.28
7.20
6.86
5.40
10.50
5.41
27.90
7.27
6.06
5.11

Erythropoietin receptor
Tumor Necrosis factor (ligand) superfamilyInterferon regulatory factor 7
Mast cell protease 8
Cathepsin E
MMP-8*Caspase 8
Cytochrome c oxidase
Dipeptidylpeptidase 4
Hepatocyte growth factor inhibitor 1
Calmodulin 3
Phosphatidylinositol-4-phosphate 5-kinase
Cyclin-dependent kinase inhibitor 3
G protein-coupled receptor kinase 6
Protein kinase C. 0
Mitogen activated protein kinase 1
Proteoglycan 2, bone marrow
LectinCadherin 1
Integrin ␤7
Tubulin ␤2
GATA binding protein 1
Calmodulin binding protein 1
Fc receptor, IgE
Leptin receptor
Cyclin B1
Cyclin E
CD47 antigen

NM_017002
BF286267
BF411036
NM_021598
NM_012938
NM_022221
NM_022277
X15030J02997
BI280343
NM_012518
BF406008
BE113362
NM_031657
AI171093
BI282894
NM_031619
NM_012976
NM_031334
AF003598
AA892044
NM_012764
AW528001
M17153
AF304191
L11995AW913890
NM_019195

0.112
0.161
0.179
0.011
0.070
0.093
0.104
0.156
0.156
0.151
0.100
0.126
0.136
0.138
0.148
0.184
0.052
0.118
0.122
0.157
0.174
0.080
0.186
0.029
0.056
0.061
0.086
0.104

*Confirmed by qRT-PCR.

ment of periapical periodontitis (8). Increased numbers of inflammatory cells or high levels of inflammatory mediators such as cytokines andproteases have been reported in periapical lesions (9). Thus, several in
vitro studies may contribute to clarify the pathophysiology of periapicallesions that lead to periapical healing (9 –11), but conclusive evaluations rely on clinical examination in patients with periapical lesions. In
fact, the radiographic evaluation is frequently used for the assessment of
RCT or even epidemiologic clinical studies (12, 13). However, radiographic examination is not sufficient for the evaluation of healing during

JOE — Volume 33, Number 8, August 2007

RCT because bone remodeling is much slower than biological responses; therefore several molecular markers must be selected for
diagnosis of periapical healing to evaluate more scientifically and reasonably. In this case we may use exudate as samples for checking several molecular markers.
At first, to identify candidate genes regulated by performing RCT
comprehensively, we established the in vivo models for periapical lesions with RCT and confirmed the inflammation and healing phaseduring RCT radiographically and histologically (Fig. 1B). Histological

Gene Profiles during Root Canal Treatment939

Basic Research—Biology
TABLE 2. Gene Profiles in Healing Phase after Root Canal Filling Compared with Healthy Control
Function
Up-regulated (total 410 genes)
Cytokine-related

Enzymes

Growth factor–related
Cellular matrix
Transcription regulator
OthersDown-regulated (total 826 genes)
Enzymes

Kinases
Cellular matrix
Transcription regulator
OthersGene Name

Accession #Fold Change

IL-1␤
Interferon regulatory factor 6
IL-1␣
Chemokine (C-C motif) ligand 2
Chemokine (C-C motif) ligand 3
MMP-3
Prostaglandin E synthase
MMP-10
Lysil Oxidase
Plasminogen activator, urokinase*Cathepsin L
MMP-14
TIMP-1Cathepsin C
PDGF-␣
Epidermal growth factor receptor
Fibroblast growth factor-3
Integrin ␤6
Collagen type V ␣3
Procollagen, type XII, ␣1
Hypoxia inducible factor 1, ␣ subunit
CCAAT/enhancer binding protein, ␤
JUN-B oncogene
Claudin 1
Heat shock 70kD protein 1A
Heat shock 27kD protein 1

NM_031512
BF410603
NM_017019
NM_031530
U22414
NM_133523
AB048730
NM_133514
NM_017061
X83537
AI176595
X83537
NM_053819
AW920064
AA866419
M37394
BF410980
AI070686
NM_021760
BE108345
AI029318
NM_024125
NM_021836
AI137640
NM_031971
NM_031970

21.9
19.6
19.3
15.5
12.9
17.4
14.8
11.9
11.5
11.3
9.95
9.85
7.73
5.85
18.2
13.7
5.27
59.1
27.4
11.2
14.4
8.26
7.65
21.6
20.0
20.0

Mast cell protease 9
MMP-8
Cathepsin E
Chymase 1, mast cell
Dipeptidylpeptidase 4
Protein kinase C, ␪
MMP-14
Proteoglycan 2, bone marrow
Tubulin ␤5
Integrin ␤7
GATA binding protein 1
CCAAT/enhancer binding protein, ␧
GATA binding protein 2
Defensin NP-4 precursor 1*Defensin, ␣5, Paneth cell-specificDefensin Rat NP-3 precursor
Cathelicidin
Cyclin-dependent kinase inhibitor 3
Cyclin A2
Cyclin B1
Cyclin E
Fc receptor, IgE, high affinity I
Leptin receptor
Cyclin D3

NM_019323
NM_022221
NM_012938
NM_013092
J02997
AI171083
NM_031020
NM_031619
AB011679
AF003598
NM_012764
NM_017095
NM_033442
U16684
U16686
U16683
AA998531
BE113362
AA998516
L11995AW913890
M17153
AF304191
NM_012766

0.01
0.01
0.08
0.09
0.13
0.15
0.18
0.01
0.15
0.20
0.01
0.06
0.13
0.01
0.01
0.01
0.02
0.07
0.07
0.07
0.08
0.10
0.10
0.17

*Confirmed by qRT-PCR.

examinations of the periapical lesions show the increased infiltration of
inflammatory cells such as leukocytes at 4 wk after pulp exposure although they were present in a small number (inflammation phase).
Further, we observed regenerative bone slightly at 4 wk after RCF (healing phase). Importantly, radiolucent areas have been still observed atperiapical lesions at 4 wk after RCF, although the areas reduced byradiographic examination. These findings indicate that periapical lesions at 4 wk after RCF were in the initial phase toward healing. Using
this in vivo model, we performed cDNA microarray analysis to clarify theregulation of gene expression comprehensively at periapical lesions
with RCT.
Microarray data demonstrates that the expression of several inflammatory cytokine-related genes is altered by inflammatory process.
After 4 wk exposure of pulp, we observed that expression of chemokine
940

Arias Martinez et al.

(CXC motif) ligand 1, IL-1␤, and colony stimulating factor 1 genes
increased as expected. Interestingly, gene expression of TNF family decreased. Since different expression patterns were observed, even in
genes categorized in similar function, we recognized that gene expression profiles are complex. To determine the biologically relevant networks and pathways of the differentially expressed genes, pathway analysis was done on the up- and down-regulated genes, and integrated total
datasets using the IPA software (supplementary Figs. 1–3). The networks describe functional relationships between gene transcripts basedon known interactions reported in the literature. Several significant
pathways were recognized in up- and down-regulated genes. Since the
network was complex as expected, we cannot find an important pathway
especially in periapical lesions during RCT. The whole extent of gene
expression and interaction cannot entirely be discussed herein; how-

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Basic Research—Biology
TABLE 3. Gene Profiles in Healing Phase after Root Canal Filling Compared with Inflammatory Phase
Function
Up-regulated (total 133 genes)
Cytokine-related
Enzymes

Cellular matrix
OthersDown-regulated (total 50 genes)
Enzymes
Cellular matrix

Transcription regulator
OthersGene Name

Accession #Fold Change*IL-1␣
Interferon regulatory factor 6
Carbonic anhydrase 4
MMP-12
Lipase, endothelial
Adenosine deaminase
MMP-3
Interferon gamma induced GTPase
Integrin ␤6
Claudin 4
Immunoglobulin heavy chain 1a
PERP, TP53 apoptosis efector
GO/G1 switch gene 2
Annexin A8
Fc receptor, IgG, low affinity lib

NM_017019
BF410603
NM_019174
NM_053963
AA964219
NM_130399
NM_133523
AW525366
AI070686
BE328951
AI411947
BI286396
AI406939
BM389254
X73371

7.15
5.67
17.4
16.9
10.5
8.42
7.90
5.73
40.5
85.4
14.1
12.5
9.48
8.95
6.13

Carbonic anhydrase 1
Pre-eosinophil-associated ribonuclease-2
Mast cell protease 9
Proteglycan 2, bone marrow
Spectrin ␣1
Myosin VC
Claudin 5
Lectin, galactose binding
Nucleotide binding protein 2
Defensin NP-4 precursor
Defensin NP-3 precursor*Defensin, ␣5, Paneth cell-specific
Cathelicidin
Transferrin

BM383006
AI177934
NM_019323
NM_031619
AI639523
BF420807
BI281680
NM_012976
BF397271
U16684
U16683
U16686
AA998531
AA945178

0.015
0.066
0.114
0.013
0.021
0.126
0.180
0.191
0.050
0.010
0.010
0.010
0.079
0.136

*Confirmed by qRT-PCR.

ever, additional investigations can be based on the data recruited by thisanalysis. We hope that they will help future investigation of molecularmechanisms in healing process after RCT.
By comparing the gene profiles between inflammation and healing
phase we focused on the IL-1 gene expression mainly in the events
associated with RCT. IL-1 is a pro-inflammatory cytokine that regulates
multiple aspects of immune and inflammatory responses (14). The IL-1
family of cytokines includes IL-1␣ and IL-1␤ (15). Since IL-1␣ and

Figure 2. Validation of microarray gene expression data by qRT-PCR analysis.
Based on the microarray data shown in Table 1–3, several genes were selected
and confirmed the expression pattern by triplicate qRT-PCR analysis for eachpooled RNA sample. The expression patterns of all the genes indicated withasterisks except caspase 8 (D), correlated to microarray data. (A): IL-1␤
(Healthy vs Inflammation), (B): Cathepsin L (Healthy vs Healing), (C): IL-1␣
(Inflammation vs Healing), (D): Caspase 8 (Healthy vs Inflammation), (E):
Defensin␣5 (Healthy vs Healing), and (F): Defensin␣5 (Inflammation vs
Healing). All values were normalized to ␤-actin housekeeping genes. Values
are mean ⫾ SD, (n ⫽ 3) * p ⬍ 0.05; ** p ⬍ 0.01; NS, not significant (Student’s
t test).

JOE — Volume 33, Number 8, August 2007

IL-1␤ bind to the same receptors (16), it seems that differential expression of these molecules may explain the differential roles of these molecules among inflammatory reactions during RCT. Interestingly, Matsuo
et al. reported previously that the exudates from the canals with small
radiolucent areas contained significantly higher IL-1␣ levels than thosefrom the canals with large radiolucent areas (17). They mentioned thatthe tendency to be an increase in the levels of IL-1␣ and a decrease in
the levels of IL-1␤ was observed after RCT. Their findings suggest that
IL-1␣ and IL-1␤ may play different roles in the healing process of
periapical lesions during RCT. Corresponding to their findings, ourresults showed that IL-1␣ expression in healing phase is stronger thanthat of inflammation phase from our cDNA microarray analysis (Table 3)
and IHC data (Fig. 3). In addition, Egusa et al. reported that humangingival epithelial cells clearly increased production of IL-1␣ in response to bacterial infection. This report might indicate that IL-1␣ level
increases by growth of epithelial cells around periapical lesion as seenin healing phase (18). It might be explained, at least in part, the pathophysiology of periapical healing by the different expression of IL-1␣ andIL-1␤, although the exact mechanisms for the functional discrimination
between IL-1␣ and IL-1␤ remains to be elucidated.
Neutrophils are key components of innate immune system (19).
They are the first cells to infiltrate at sites of local inflamed lesions where
they kill invading pathogens and promote acute inflammation via release of proteases and reactive oxygen species (19, 20). In our experimental in vivo model, we observed that leukocytes infiltrate at periapical lesions in inflammation phase. These data suggest that neutrophils
are important for the initiation of periapical lesion formation. Furthermore, it has been generally accepted that neutrophils produced antimicrobial peptides defensins (21), suggesting that neutrophils can protect
against severe inflammation and its potential pathological conse-

Gene Profiles during Root Canal Treatment

941

Basic Research—Biology

Figure 3. Immunohistochemical validation of gene expressions obtained from microarray data in periapical tissues. IL-1␣ (A–H) and IL-1␤ (I–P). Immunohistochemistry was performed by an indirect peroxidase technique described in “Materials and Methods” section. From each group 3 sections were stained on a slide for
either IL-1␣ and IL-1␤, and the most representative staining was selected. Positivity is indicated by a brown color. Negative controls (without the first antibody), A,
E, I, M. Scale bars ⫽ 500 ␮m for A–D, I–L; 100 ␮m for E–H, M–P.

quences. Mainly, defensins have been isolated from the cytoplasmic
granules of neutrophils in human beings (22), rats (23), and rabbits(24). From the present microarray data, we found that the expression ofseveral defensin-related genes, defensin NP-4 precursor, defensin Rat
NP-3 precursor and defensin ␣5, were down-regulated at healing phasecompared with inflammation phase (Table 3). These findings suggestthat infiltration of neutrophils at periapical lesions decreased after RCF.
To examine the expression of defensin-related genes in periapical lesions might be useful to create the novel examination systems for diagnosis of periapical healing after RCT in the future.
In summary, cDNA microarray analysis of global gene expression patterns provided new molecular candidates for the diagnosisof periapical healing after RCT using in vivo models. Our findings
suggest biological significance of differential gene expression andconfirm some results in the literature with regard to molecules
known to be involved in the periapical healing. cDNA microarray
analysis of a well-controlled model system involving a highly relevant periapical healing is a useful screening tool for identifying
novel differentially expressed genes. These genes provide a basis forfurther studies of periapical healing after RCT, and might be usefulas attractive targets for diagnosis.

Acknowledgments
We thank Dr. K. Yamabe and Dr. O. Rodis for technical support.
This study was supported by Grants-in-Aid for Scientific Research(A) (No. 16209056, No. 18209057, and No. 16209063) from theJapan Society for the Promotion of Science.

Supplementary Material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.joen.2007.04.016.
942

Arias Martinez et al.

References
1. Torabinejad M, Eby WC, Naidorf IJ. Inflammatory and immunological aspects of thepathogenesis of human periapical lesions. J Endod 1985;11:479 – 8.
2. Marton IJ, Kiss C. Protective and destructive immune reactions in apical periodontitis. Oral Microbiol Immunol 2000;15:139 –50.
3. Oztan MD. Endodontic treatment of teeth associated with a large periapical lesion. Int
Endod J 2002;35:73– 8.
4. Mikrogeorgis G, Lyroudia K, Molyvdas I, Nikolaidis N, Pitas I. Digital radiographregistration and subtraction: a useful tool for the evaluation of the progress ofchronic apical periodontitis. J Endod 2004;30:513–7.
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6. Dalma-Weiszhausz DD, Warrington J, Tanimoto EY, Miyada CG. The affymetrix
GeneChip platform: an overview. Methods Enzymol 2006;410:3–28.
7. Fouad AF. IL-1 alpha and TNF-alpha expression in early periapical lesions of normal
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9. Danin J, Linder LE, Lundqvist G, Andersson L. Tumor necrosis factor-alpha andtransforming growth factor-beta1 in chronic periapical lesions. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2000;90:514 –7.
10. Lee SH, Kim KK, Choi BK. Upregulation of intercellular adhesion molecule 1 andproinflammatory cytokines by the major surface proteins of Treponema maltophilum and Treponema lecithinolyticum, the phylogenetic group IV oral spirochetes
associated with periodontitis and endodontic infections. Infect Immun 2005;
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2003;96:492– 8.
12. Kirkevang LL, Wenzel A. Risk indicators for apical periodontitis. Community Dent
Oral Epidemiol 2003;31:59 – 67.
13. Kabak Y, Abbott PV. Prevalence of apical periodontitis and the quality of endodontic treatment in an adult Belarusian population. Int Endod J 2005;38:
238 – 45.
14. Seymour GJ. Possible mechanisms involved in the immunoregulation of chronic
inflammatory periodontal disease. J Dent Res 1987;66:2–9.

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15. Rosenwasser LJ. Biologic activities of IL-1 and its role in human disease. J Allergy Clin
Immunol 1998;102:344 –50.
16. Boch JA, Wara-aswapati N, Auron PE. Interleukin 1 signal transduction— current
concepts and relevance to periodontitis. J Dent Res 2001;80:400 –7.
17. Matsuo T, Ebisu S, Nakanishi T, Yonemura K, Harada Y, Okada H. IL-1alpha andIL-1beta periapical exudates of infected root canals: correlations with the clinicalfindings of the involved teeth. J Endod 1994;20:432–5.
18. Egusa H, Nikawa H, Makihira S, Yatani H, Hamada T. In vitro mechanisms of interleukin-8-mediated responses of human gingival epithelial cells to Candida albicans
infection. Int J Med Microbiol 2006;296:301–11.
19. Mortensen RF, Zhong W. Regulation of phagocytic leukocyte activities by C-reactive
protein. J Leukoc Biol 2000;67:495–500.

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20. Kadl A, Bochkov VN, Huber J, Leitinger N. Apoptotic cells as sources for biologically
active oxidized phospholipids. Antioxid Redox Signal 2004;6:311–20.
21. McGirt LY, Beck LA. Innate immune defects in atopic dermatitis. J Allergy Clin Immunol 2006;118:202– 8.
22. Wilde CG, Griffith JE, Marra MN, Snable JL, Scott RW. Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family. J Biol
Chem 1989;264:11200 –3.
23. Eisenhauer PB, Harwig SS, Szklarek D, Ganz T, Selsted ME, Lehrer RI. Purification
and antimicrobial properties of three defensins from rat neutrophils. Infect Immun
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24. Selsted ME, Harwig SS, Ganz T, Schilling JW, Lehrer RI. Primary structures of threehuman neutrophil defensins. J Clin Invest 1985;76:1436 –9.

Gene Profiles during Root Canal Treatment

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The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

Oligonucleotide array analysis of cyclic tension-responsive
genes in human periodontal ligament fibroblasts
Keisuke Yamashiro a , Fumio Myokai a , Koichi Hiratsuka b , Tadashi Yamamoto a ,Kyoko Senoo a , Hideo Arai a , Fusanori Nishimura a,1 ,Yoshimitsu Abiko b , Shogo Takashiba a,∗a Department of Pathophysiology–Periodontal Science, Okayama University Graduate School of Medicine,

Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8525, Japanb Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo,

2-870-1 Sakae-cho-Nishi Matsudo, Chiba 271-8587, Japan
Received 19 September 2006; received in revised form 7 December 2006; accepted 2 January 2007
Available online 23 January 2007

Abstract
Mechanical stress results in differential gene expression that is critical to convert the stimulus into biochemical signals. Under
physiological stress such as occlusal force, human periodontal ligament fibroblasts (HPLF) are associated with homeostasis ofperiodontal tissues however the changes in response to mechanotransduction remain uncharacterized. We hypothesized that cyclic
tension-responsive (CT) genes may be used to identify a set of fundamental pathways of mechanotransduction. Our goal was tocatalogue CT genes in cultured HPLF. HPLF were subjected to cyclic tension up to 16 h, and total RNA was isolated from bothtension-loaded and static HPLF. The oligonucleotide arrays analysis revealed significant changes of mRNA accumulation for 122
CT genes, and their kinetics were assigned by the K-means clustering methods. Ingenuity Pathway Analysis was completed for
HPLF mechanotransduction using 50 CT genes. This analysis revealed that cyclic tension immediately down-regulated all nuclear
transcription factors except v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) reacting as an early responsive gene. Inturn, transcription factors such as tumor protein p53 binding protein 2 (TP53BP2), and extra-nuclear molecules such as adrenergic
receptor β2 (ADRB2) were up-regulated after 1–2 h, which may result in fundamental HPLF functions to adapt to cyclic tension.
Subsequent inhibition assays using Y27632, a pharmacologic inhibitor of Rho-associated kinase (ROCK), suggested that HPLF
has both ROCK-dependent and ROCK-independent CT genes. Mechanical stress was found to effect the expression of numerous
genes, in particular, expression of an early responsive gene; FOS initiates alteration of HPLF behaviors to control homeostasis of
the periodontal ligament.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Cyclic tension-responsive genes; Oligonucleotide array; Human periodontal ligament fibroblasts; Pathway analysis; Rho-associatedkinase∗ Corresponding author. Tel.: +81 86 235 6677;
fax: +81 86 235 6679.
E-mail address: stakashi@cc.okayama-u.ac.jp (S. Takashiba).
1 Present address: Department of Dental Science for Health Promotion, Division of Cervico-Gnathostomatology, Hiroshima University
Graduate School of Biomedical Sciences, 1-2-3 Kasumi Minamiku,
Hiroshima 743-0037, Japan

1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2007.01.015

1. Introduction
Mechanical stress is thought to play important roles
in regulating the function, metabolism or maintenanceof connective tissues such as periodontal ligaments.
Abnormal mechanical stress may cause destruction of

K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

tissues, and affects many pathological situations. A better understanding about cellular adaptation to changes
in mechanical stress may help us design a new strategy
in the treatment and prevention of mechanical stressrelated disorders such as trauma, tooth/bone resorption,
and atrophy or destruction of the connective tissues.
Mechanotransduction is the process by which cells
transduce physiological force-induced signals into biochemical responses, and involves differential gene
expression that is necessary for mediating adaptations
to mechanical stress (Ingber, 2003). Evidence has accumulated that mechanical stress-induced alterations incell shape and structure are critical for the control ofcell contraction, migration, growth, differentiation, andapoptosis (Chicurel, Chen, & Ingber, 1998; Khan &Sheetz, 1997). Mechanical stresses are either transmitted to cells from the extracellular matrix, or generated
within the contractile cytoskeleton of individual cells(Wang, Butler, & Ingber, 1993). Force-induced assemblyof the focal adhesions and the cytoskeleton are crucial for
inducing mechanotransduction in the cytoplasm (Geiger& Bershadsky, 2001; Ingber, 1997), and mediated inlarge by activation of the Ras-homolog of small GTPase(Rho) (Amano et al., 1997; Ridley & Hall, 1992). Rhoacts as a molecular switch that cycles between an inactive GDP-bound and an active GTP-bound conformationinteracting with downstream targets (effectors) to elicit
cellular responses, and regulate the actin cytoskeleton (Etienne-Manneville & Hall, 2002). Rho-associatedkinases (ROCK), the first and the best-characterized Rho
effectors, are serine/threonine kinases that are involved
in diverse cellular functions including motility, adhesion, smooth-muscle contraction, neuritic outgrowth,
centrosome positioning, apoptosis, actin cytoskeleton
organization and cell-size regulation. Thus, ROCK havethe potential to contribute to various physiologicaland pathological states (Maekawa et al., 1999; Riento& Ridley, 2003). The Rho/ROCK pathway has beenshown to be crucial for mediating mechanotransduction(Chiquet, Renedo, Huber, & Fluck, 2003; Numaguchi,
Eguchi, Yamakawa, Motley, & Inagami, 1999) however the precise mechanisms that link mechanical stressto subsequent changes in cellular activities remains
unknown.
Periodontal ligament is a connective tissue lying
between alveolar bone and tooth root, and not only isa source of osteogenic cells, but also may contain multipotent stem cells that could regenerate cementum andperiodontal ligament (Seo et al., 2004). Periodontal ligament fibroblasts (HPLF), in particular, can differentiate
in response to a variety of extracellular stimuli to either
maintain homeostasis or participate in remodeling of the911

periodontal ligament, and repair or regeneration of thesurrounding hard tissues (McCulloch, Lekic, & McKee,2000). These cells are subject to continuous mechanicalstress under physiological condition the result of both
occlusal and masticatory forces. Several studies havebeen published that link mechanical stress to intracellular signaling molecules in HPLF (Kletsas, Basdra, &Papavassiliou, 2002; Myokai et al., 2003) however themolecular mechanisms of HPLF mechanotransduction
remain elusive. Since it is likely that the expression ofmany genes is altered in response to mechanical stress it
will become necessary to further catalogue these genes
with respect to both temporal and subcellular distribution. We hypothesized that comprehensive analysis of
cyclic tension-responsive (CT) genes may provide data
useful to understand the fundamental mechanisms of
mechanotransduction in HPLF.
The GeneChip probe array technology allows asystematic analysis of gene expression changes, and provides significant information for a wide variety of basic
biological processes, including development (Abeyta
et al., 2004), tumorigenesis (Kasamatsu et al., 2005),
and the immune system (Calvano et al., 2005). The
array technology allows simultaneous monitoring of theactivities of numerous genes because of the technique’s
sensitivity, specificity, and reproducibility. In the present
study, HPLF were loaded with cyclic tension, and the CT
genes were identified using the GeneChip probe array.
The CT genes identified were further analyzed on acellular pathway map based on Ingenuity Systems’ Pathway Knowledge Base. This was done to identify genetic
networks involved in mechanotransduction in HPLF.
Moreover, to elucidate the effect of the Rho/ROCK pathway on CT gene expression, we analyzed the CT gene
expression in the presence or absence of a pharmacologic
inhibitor of ROCK activity.
2. Materials and methods
2.1. Cell culture and mechanical stress
Periodontal ligament was obtained from periodontally healthy and non-carious teeth extracted fororthodontic reasons from four donors, three Japanese
female (21, 24, and 17 years old) and a Japanese male
(22 years old), with informed consent. Prior to the experiment, the protocol (no. 43) was approved by the Research
Ethics Committee for Human Genome/Gene Analysis Research in Okayama University Graduate Schoolof Medicine, Dentistry, and Pharmaceutical Sciences.
HPLF were maintained and expanded in Dulbecco’s
Modified Eagle’s Medium supplemented with 10% fetal

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K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

calf serum (both reagents from Invitrogen, Carlsbad,
CA) as described previously (Yamamoto et al., 2003).
HPLF between the 5th and 9th passages were seeded on
6-well flexible-bottomed culture plates (FLEX II; Flexcell, McKeesport, PA) at a concentration of 2 × 105 cells
per well, and grown for 2 days to allow them to attach on
the culture dish while the control cells were seeded on
6-well fix-bottomed culture plates (FLEX I; Flexcell).
The HPLF grown on the flexible-bottomed plates
were subjected to 18% tension (36 kPa) at 6 cycles per
minute with the Flexercell Strain Unit for 0.5, 1, 2 or 16 h
as described (Myokai et al., 2003). It has been demonstrated that the degree of tension can be calculated after
measuring the increase in distance between any two setsof concentric rings of the culture well (Banes, Link,
Gilbert, Tay, & Monbureau, 1990).
The Flexercell Strain Unit is designed to apply reproducible cyclic tension or compression to the cells in vitro.
Force Analysis showed that deformation of the flexible
membrane is not uniform but rather generates a tension
gradient with the greatest deformation occurring at the
periphery of the well (Banes et al., 1990). Thus, theresults represented an average of cells exposed to different degrees of tension. The calculation showed that 18%
tension resulted in 6% mean elongation to cells over theentire culture plate surface. Control cells were cultured
under identical conditions but remained static.
2.2. Oligonucleotide microarray analysis
The oligonucleotide microarray analysis was performed on HPLF at 5 different time points (0, 0.5,
1.0, 2.0, and 16 h) of cyclic tension. HPLF from threedonors were used for these analyses. Total RNA wasrecovered from the tension-loaded and control HPLF
using RNeasy Mini Kit (Qiagen, Hilden, Germany). The
protocol for microarray analysis was described previously (Hiratsuka et al., 2002). Briefly, single-stranded(ss) cDNA was synthesized from 8 ␮g of total RNA
using a T7-(dT)24 primer (Amersham Biosciences, Piscataway, NJ) with SuperScript II Reverse Transcriptase(Invitrogen). Double-stranded (ds) cDNA was synthesized from the sscDNA, and purified with the GeneChip
Sample Cleanup Module (Affymetrix, Santa Clara, CA).
Using the dscDNA as a template, cRNA was transcribedwith Bio-11-CTP and Bio-16-UTP (Enzo Diagnostics,
Farmingdale, NY), purified, fragmented, and hybridized
to the array (Human Genome Focus GeneChip probearrays #900377; Affymetrix) spotted about 8500 known
human genes. The arrays were washed, and stained withstreptavidin–phycoerythrin (Molecular Probes, Eugene,OR) using an Affymetrix Fluidics Station. Fluorescenceintensities were scanned with an Affymetrix GeneArray
Scanner.
Poly-A RNA controls were used to monitor theentire target labeling process. The GeneChip Poly-A
RNA Control Kit (Affymetrix; P/N 900433) contained
a pre-synthesized mixture of lys, phe, thr, and dap
from Bacillus subtilis. The final concentrations of thecontrols, relative to the total RNA population, were
1:1 × 106 , 1:5 × 104 , 1:2.5 × 104 , 1:7.5 × 103 , respectively. Biotin-labeled cRNAs were used to evaluate
sample hybridization efficiency on the microarrays.
The GeneChip Eukaryotic Hybridization Control Kit(Affymetrix; P/N 900299 and 900362) contained 20×
eukaryotic hybridization controls that were composedof a mixture of biotin-labeled cRNA transcripts of bioB,
bioC, bioD, and cre, prepared in staggered concentrations (1.5, 5, 25, and 100 pM final concentrations
for bioB, bioC, bioD, and cre, respectively). The 20×controls were spiked into the hybridization cocktail independent of RNA sample.
2.3. Data analysis
Data analysis was performed as described(Rajagopalan, 2003) using the Affymetrix Microarray
Suite software (version 5.0, Affymetrix) and GeneSpring software (version 7.1; Tomy Digital Biology,
Tokyo, Japan). In brief, the hybridization efficiencies
were equalized by median value of the control probe
set to each array, and the genes with higher signal
intensities than bioB were selected for further analysis.
The microarrays are divided into several hundred
thousand probe cells grouped in pairs, perfect match(PM) and mis-match (MM) cells. Therefore, detection cells were made on the basis of a discrimination
score, Ri which was defined for each probe pair as
Ri = (PMi − MMi )/(PMi + MMi ), where PMi and MMi
were the intensity of the perfect match and mis-match
cells of probe pair i, respectively.
To assess the extent of variation accumulated during
sample preparation, hybridization, and scanning procedures, raw intensity values from two different probe chiparrays were plotted against each other on a log scatter
plot: the intensity of tension-free HPLF on the horizontal axis; that of tension-loaded HPLF, at 0.5, 1.0, 2.0 or
16 h, on the vertical axis. To confirm the microarray data,real-time reverse transcription-polymerase reaction (RTPCR) was performed as described below for randomly
selected genes.
Global normalization was performed for dataobtained from the raw data. The normalized data
were analyzed statistically by one-way ANOVAK. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921(p < 0.05) and K-means clustering methods (n = 8).
The functional annotation of the CT genes were
examined using the Agilent Databases (Agilent Technologies, Palo Alto, CA) and the Affymetrix Databases(http://www.affymetrix.com/analysis/netaffx/xmlquery.
affx). The CT genes were classified into categories
based on the biological process in the GeneSpring Gene
Ontology (Agilent).
Ingenuity Systems’ Pathways Knowledge Base (version 3.0: Winter’04 Release containing 20,000 genes;
Ingenuity Systems, Mountain View, CA) was used tosearch for cellular pathways affected by cyclic tension.
A detailed description of ingenuity pathways analysis (IPA) can be found on http://www.Ingenuity.com.
Data sets containing CT genes accession numbers andtheir corresponding expression fold-change values were
imported into IPA software. Focus genes identified by the
IPA program were categorized based on location, cellular components, and reported biochemical, biologic, andmolecular functions using the software. The program
queried the Ingenuity Systems’ Pathways Knowledge
Base for interactions between the focus genes and allother gene objects stored in the knowledge base, andgenerated a set of networks ranked by score. The scoreis the negative log of a p value and indicates the likelihood of the focus genes being found in the networks due
to random chance. A score of 3 indicates that there is a
1/1000 chance that the focus genes are in a network duesolely to chance. Therefore, scores of 3 or higher represent a 99.9% confidence level. Biological functions werethen calculated and assigned to each network.
2.4. Phalloidin staining
Static HPLF were cultured and either treated with
1 ␮M Y27632 or treated with solvent alone (Cat. 688000;
CALBIOCHEM, San Diego, CA) for 1, 2, 8 or 16 h.
To detect F-actin fibers, HPLF were fixed with 3.7%
formaldehyde, washed twice with PBS (pH 7.2) supplemented with 0.5% Triton X-100, and treated with
5 mg/ml of Texas Red-X phalloidin (Cat. T-7471; Molecular Probes) for 2 h. Staining signals were visualizedusing a fluorescence microscope (BX50; Olympus,
Tokyo, Japan), and images captured with a digital camera(DP70; Olympus).
2.5. Quantitative mRNA analysis of HPLF treatedwith Y27632
HPLF from a donor (24-year-old female) werepre-incubated with or without Y27632 for 1 h, andloaded with cyclic tension for different periods (0, 0.5,913

1.0, 2.0 or 16 h). We randomly selected several CT genes
in a merged genetic network, and performed real-time
RT-PCR analysis. One microgram of total RNA wasrecovered from the tension-loaded and tension-free
HPLF, and reverse-transcribed with oligo (dT)12–18
primer, dNTPs, and SuperScript II (all from Invitrogen)in a total volume of 20 ␮l. One microliter of thisreaction was mixed with QuantiTect SYBR Green PCR
Master Mix (Qiagen) and the gene-specific primers
designed by Primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3 www.cgi). Real-time RT-PCR was
performed using the DNA Engine Opticon 2 (Bio-Rad,
Hercules, CA) according to the manufacturer’s instructions. The amplification conditions consisted of an
initial 15 min denaturation step at 95 ◦ C, followed by
40 cycles of denaturation at 94 ◦ C for 15 s, annealingat 55 ◦ C for 30 s, and elongation at 72 ◦ C for 30 s. Four
independent assays were performed for each gene.
Relative expression was shown after normalization
against the expression for ␤-actin mRNA. The data
were analyzed statistically by Student’s t-test. Values ofp < 0.05 were considered significant.
3. Results
3.1. Identiﬁcation and characterization of CT genes
in HPLF
Microarray analysis was performed using HPLF subjected to cyclic tension for up to 16 h and control HPLF. A
considerably higher number of transcripts exhibited signal intensities differing by >2-fold with tension-loaded
versus control HPLF in the replicates. Each result wasrepresented in log/log scatter plots to evaluate the consistency of the data. The microarray data was confirmed
using real-time RT-PCR on randomly selected gene transcripts (data not shown).
We performed global normalization on the microarray data from all three donors, and identified 122 CT
genes that had levels of expression significantly alteredby the cyclic tension (Supplementary-1). To visualize
the overall response to cyclic tension, kinetic patternsof the CT transcripts were analyzed with the K-meansclustering method, and the all CT genes were assigned
to eight clusters (Fig. 1, Supplementary-1). The clusters 2, 5, 6 and 7 included tension-induced genes while
the clusters 3 and 4 included tension-suppressed genes.
As a functional classification of CT genes, a list ofseven different categories was established based on
GeneSpring Database (Supplementary-2). All clusters
included genes that could be included in two categories:
intracellular signaling and cell growth/maintenance.

914

K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

Fig. 1. K-means clustering of CT genes. The 122 CT genes were assigned to 8 clusters by applying K-means methods. Each cluster is represented
by the centroid (average pattern) of the kinetics. The number of clusters and assigned genes is indicated on the top of each kinetic graph. The x
axis represents application periods of cyclic tension (0, 0.5, 1.0, 2.0, or 16 h), and the y axis represents the expression value, indicated after global
normalization, in logarithmical scale.

Both of these categories are composed of fundamental
biological processes such as metabolism, biogenesis, cell
cycle, cell growth, and cell death.
3.2. Identiﬁcation of genetic networks affected bycyclic tension on HPLF
To examine how CT genes interact through perturbed
biological networks, we performed network analysis
on CT genes using Ingenuity Pathway Analysis (IPA).
IPA provides a tool to construct and extract relevant
information from the microarray analysis, and integrates
individual genes. Of the 122 CT genes, 63 were mapped
to genetic networks defined by IPA. These networks
describe functional relationships among gene products
based on findings presented in peer-reviewed scientificpublications. IPA associated these networks with known
biological pathways, and identified as significant 24
networks. Notably, the top 4 genetic networks with ascore of 10 or higher and containing 10 or more focus
genes represent the extensive interrelation and interaction between the genes affected by cyclic tension, which
regulate unique biological processes (Table 1). Since
overlapping genes were not detected in network-4, three
networks were merged to form a major network containing 50 focus genes regulating cell death, cellularassembly and organization, and cellular function andmaintenance (Table 1, Fig. 2, Supplementary-3). The
merged network included key mediators, such as Bcell CLL/lymphoma 2 (BCL2) and v-fos FBJ murine
osteosarcoma viral oncogene homolog (FOS). For abetter understanding of the temporal and spatial gene
expression in response to mechanotransduction, we per-formed sequential analysis using the merged pathway(Fig. 2, Supplemantary-3). The analysis revealed thatthe cyclic tension induced a transient and self-limiting
response based on alterations in RNA abundance andfunctional subcellular localization. After 0.5 h of cyclic
tension, all nuclear transcription factors were downregulated except FOS, which reached a maximum levelas an early responsive gene. The nuclear transcription
factors such as tumor protein p53 binding protein 2(TP53BP2), and TGFβ inducible early growth response(TIEG), and extra-nucleic molecules such as adrenergic receptor β2 (ADRB2) and immediate early response(IER3) were up-regulated 1–2 h after cyclic tension,
apparently a critical event to adapt to external changes
resulting from mechanical stress (Fig. 2, Supplementary3). After 16 h of cyclic tension, the cells subjected tomechanotransduction returned to homeostasis (0 h). We
identified correlations among these results through theuse of the K-means cluster (Fig. 1). While early responsive genes, FOS and BCL2 were categorized in cluster
2, all 14 tension-suppressed genes were localized in the
nucleus and categorized in cluster 3 or 4. Clusters 5,
6 and 7 included 15 tension-induced genes that weremainly localized outside the nucleus (Fig. 3).
3.3. Effect of Rho/ROCK pathway on CT gene
expression in HPLF
To examine how the Rho/ROCK pathway participates in regulating mechanotransduction in HPLF, weanalyzed the kinetics of selected CT genes in the presence or absence of Y27632, a small-molecule inhibitorof ROCK activity (Uehata et al., 1997). It has been shown

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915

Fig. 2. Pathway analysis of focus CT genes involved in mechanotransduction. A prototypical pathway in a tension-loaded cell was constructedfrom 50 focus CT genes involved in mechanotransduction. Temporal and spatial changes of the expression of CT genes were analyzed at different
time periods of cyclic tension (0, 0.5, 1.0, 2.0, or 16 h), and the pathway after 1 h of stressing was shown (see also Supplementary-3). Genes
statistically increased from baseline are colored red while decreased are green. Bold genes were identified as CT genes by the microarray analysis,
and are listed in Table 1. Other genes were neither on the expression array nor significantly regulated. Abbreviations of these genes can be found in
Supplementary-4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

Table 1
Genetic networks affected by cyclic tension
Network ID

Focus genesa

Scoreb

Top functions

1

ACY1, ADRB2, APPBP1, AR, ATF1, BAG1, BCL2, CAMK4,
CASP3, CCNG2, CYCS, EIF2B1, EIF4G3, EMB, ETV5, FOS, GLI,
GLI2, GOSR1, HDAC3, HDAC4, HNF4A, MCF2, POLR3A,
POLR3C, POLR3F, PPP2CA, PPP2R5A, PRKR, PSMA3, RAC1,
RIPK1, SSB, TP53BP2, ZNFN1A4BGLAP, CASC3, CRADD, DGKZ, DMD, DTNA, DYSBINDIN,
E2F1, GTF2A1, GTF2A2, GTF2B, GTF2F2, HOXA11, HRMT1L1,
IER3, IL6, MAGOH, MCM6, MEIS1, MTPN, NCOA6, NUP153,
NXF1, RB1, REL, RELA, SNTB2, SNTG1, SUV39H1, TBP, TEAD1,
TIEG, TP53BP2, ZNF192, ZNF335
ADRB2, ADRBK1, AFAP, APS, ARRB1, CBL, CBLB, CDK5,
CKM, DAB1, DAO, EGFR, EPN1, EPS15, FRS2, HDAC6, HRB,
INSR, LEPR, MYOD1, OPRM1, REPS2, RUNX2, SCHIP1, SNX1,
SNX2, SNX4, SNX6, SORBS1, SPRY2, SRC, SYNCRIP, TACR1,
TWIST1, VAV3
CD34, CDKN2B, CEBPA, CENPE, CTNNA1, CTNNB1, DDIT3,
DUSP1, DUSP6, ELK1, EXO1, GJA1, GTF2I, JUP, MAPK1, MLH1,
MSH2, MSH6, MYB, MYBL2, MYC, ORC1L, ORC2L, ORC3L,
ORC4L, ORC5L, ORC6L, PIGC, PKN2, PMS2, PTMA, TNRC6,
TRIB1, YME1L1, ZNF42

35

Cancer, cell death, reproductive
system disease

22

Viral function, gene expression,
cellular assembly and organization

18

Cellular function and maintenance,amino acid metabolism,
developmental disorder

13

Cell cycle, DNA replication,
recombination, and repair, cancer

2

3

4

a Bold and underlined genes are those identified by the microarray analysis. The other genes are neither on microarray nor significantly regulated.
See Supplementary-4 for the abbreviations of genes.
b A score of >3 was considered significant (p < 0.001).

Fig. 3. Subcellular localization and K-means clustering of focus CT
genes on IPA. Fifty focus CT genes in IPA were classified based on thesubcellular localization and the K-means clustering. The x axis represents K-means clustering described in Fig. 1, and the y axis represents
the number of classified genes. The early responsive genes are classified in cluster 2. The clusters 5, 6 and 7 included tension-induced
genes while the clusters 3 and 4 included tension-suppressed genes.
The subcellular localizations are indicated as follows: nucleus, red;cytoplasm, yellow; plasma membrane and extracellular space, blue.
(For interpretation of the references to color in this figure legend, thereader is referred to the web version of the article.)that ROCK promotes both actin–myosin contraction andactin polymerization (Kimura et al., 1996; Maekawa etal., 1999). Thus, we first examined the effect of Y27632
on polymeric (F) actin, the dominant structural constituent of the cell cortex in static HPLF. Phalloidin
staining revealed that F-actin fibers appeared thinner andless dense in HPLF treated with Y27632 for 1 h, whereas
the dense fibers were observed clearly in HPLF without
Y27632 treatment (Fig. 4). The similar results were also
observed in HPLF following treatment with Y27632 for
16 h (data not shown). These findings indicated that inactivation of ROCK led to loss of stress fibers, and ROCK
activity was necessary for controlling actin cytoskeleton
organization in HPLF.
To examine the alteration of CT genes by Y27632, we
randomly selected 8 CT genes from the merged network(Fig. 2, Supplementary-3), and performed real-time RTPCR analysis on the tension-loaded HPLF cultured withor without Y27632 (Fig. 5). Y27632 effectively inhibited the tension-induced aminoacylase 1 (ACY1) genes
expression, and also maintained the basal expression levels of tension-suppressed activating transcription factor
1 (ATF1) and golgi SNAP receptor complex member
1 (GOSR1) genes. However, Y27632 did not significantly alter the other tension-induced genes; TP53BP2,
ADRB2, BCL2, FOS, and caspase 3 (CASP3). These
results suggested that mechanotransduction in HPLF

K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

917

Fig. 4. Effect of Y27632 on development of actin fibers in HPLF. F-actin was stained with Texas Red-X phalloidin. Static HPLF treated with
Y27632 exhibited faint F-actin fibers (B) whereas control HPLF had dense F-actin fibers (A). Three independent experiments were performed, anda typical set of results is shown. Bar equals 10 ␮m.

Fig. 5. Effect of Y27632 on mRNA expression of CT genes in HPLF. Real-time RT-PCR was performed using HPLF mRNA after 0.5–2 h of cyclic
tension. The relative expression was valued against ␤-actin expression as a fold increase. The data were analyzed statistically using Student’s t-test(* p < 0.05; ** p < 0.01). Error bars indicate standard deviation of three independent assays for each gene. Abbreviations of these genes and PCRprimer sequence can be found in Supplementary-4 and -5.

includes both Rho/ROCK-dependent and Rho/ROCKindependent pathways, and the alternative pathway maycompensate for the effect of the Rho/ROCK pathway on
CT gene expression.
4. Discussion
In this study, we have identified 122 CT genes from
HPLF using Human Genome Focus GeneChip probearray (Affymetrix). Recently, shear stress-responsive
genes have been examined in human umbilical vein

endothelial cells (HUVECs) and coronary artery
endothelial cells (HCAECs) using HuGene FL array(Affymetrix) (Ohura et al., 2003). In addition, ultraviolet (UV) radiation-induced genes have been detected
in human skin fibroblasts using HGU95Av2 arrays(Affymetrix) (Gentile, Latonen, & Laiho, 2003). The
distributions of their variability are similar to ours,suggesting that both UV radiation and cyclic tension
forces are moderate to elicit changes in the gene expression in the fibroblasts. Although the HuGene FL andHGU95Av2 arrays cover 39 and 70% of the 122 CT

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K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

genes, respectively, none of the CT genes were foundin the sets of either the shear stress-responsive genes, orthe UV radiation-induced genes. These findings suggestthat changes in fibroblast gene expression may be quite
dependent on the specific type of stress.
In previous studies, 50–100 N forces were generally
used as an occlusal loading (Holmes, Diaz-Arnold, &Leary, 1996; Southard, Behrents, & Tolley, 1989) probably because similar vertical forces have been measured
during chewing movements in several in vivo studies(Proffit, Fields, & Nixon, 1983; Richter, 1995). The
model, which is loaded with a vertical load of 100 Non the lower pre-molar, mimicks the effect of a moderate orthodontic force with the stresses across periodontal
ligament within 40 kPa (Toms & Eberhart, 2003). In thepresent study, we applied cyclic tension at 36 kPa to
the flexible-bottomed culture plates using the Flexercell
Strain Unit, thus the magnitude of cyclic tension in ourstudy is distributed within the physiological range. The
application of moderate orthodontic force causes bone
resorption, while the application of excessive orthodontic force results in excessive compressive force, which
induces local ischemia, tissue hyalinization, and cell
death in the periodontal ligament (Reitan & Rygh, 1994).
In a concerted remodeling process, it is the right balance
between death and renewal that decides between healthand disease. Although it seems to vary in different cell
types, one of candidates that modulate mechanical stressinduced apoptosis is caspase-3 (CASP3) (Earnshaw,
Martins, & Kaufmann, 1999). However, cyclic tension
did not significantly alter CASP3 expression in ourmodel. This is further evidence that the cyclic tension
applied mimicks physiological force.
The number of genes with altered expression may
reflect the complexity of the biologic response tomechanical loading, and provide guidance in investigating the intricate processes that take place in mechanicallystress-loaded cells. Therefore, the 122 CT genes wereclassified into seven functional categories based on the
GeneSpring Database, and their expression patterns wereassigned into eight clusters by the K-means clustering
methods. As expected, most of CT genes are involvedin various kinds of intra- and intercellular signalingmechanisms. The functional analysis and clustering ofindividual CT genes provides a means to assess overall changes and characterizations however this approachprovides fewer insights into specific biological processes
and signaling networks directly responsive to cyclic tension. Thus, we performed network analysis on CT genes
using IPA, and sequential analysis using the networkand the K-means clustering. We found that mechanotransduction in HPLF initiates induction of the early

responsive genes, FOS and BCL2 while other transcription factors were suppressed after 0.5 h of cyclic tension.
FOS is thought to have an important role in signal transduction, cell proliferation and differentiation, and bonedevelopment (Johnson, Spiegelman, & Papaioannou,
1992; Wang et al., 1992). The role of FOS during
mechanotransduction has been extensively describedin bone, and those studies showed that mechanicallyinduced bone formation is preceded by expressionof FOS (Iqbal & Zaidi, 2005; Pavalko et al., 1998;
Sadoshima, Takahashi, Jahn, & Izumo, 1992). Our study
also confirmed that FOS is a key mediator of mechanotransduction in HPLF exposed to cyclic tension. FOS
expression may initiate HPLF differentiation to regenerate periodontal ligament and surrounding hard tissues. It
has been reported that BCL2 negatively regulates both
apoptosis and autophagy, which is critical in tissue homeostasis, development and disease (Hockenbery, Nunez,
Milliman, Schreiber, & Korsmeyer, 1990; Pattingre et al.,2005). In HPLF, the immediate expression of apoptosispreventing BCL2 by cyclic tension may be involved in
the control of cell viability.
Following the induction of early responsive genes,
intra- and extra-nuclear molecules, such as TP53BP2,
TIEG, ADRB2 and IER3 were up-regulated 1–2 h after
cyclic tension. TP53BP2 was identified as an interacting protein with p53, BCL2 and nuclear factor ␬B(NF-␬B) (Iwabuchi, Bartel, Li, Marraccino, & Fields,
1994; Naumovski & Cleary, 1996; Yang et al., 1999).
TP53BP2 interacts with p53 to activate proapoptotic
events, and involvement in various biological processes
such as carcinogenesis has been shown (Mori, Okamoto,
Takahashi, Ueda, & Okamoto, 2000). Interestingly, IER3
is an apoptosis inhibitor involved in NF-␬B-mediatedcell survival (Wu, Ao, Prasad, Wu, & Schlossman,1998), and BCL2 and IER3 have been reported to prevent the TP53BP2 mediated apoptosis (Takahashi etal., 2005). Thus, selective actions of TP53BP2 with
BCL2, IER3 and NF-␬B may determine the susceptibility of cells to triggering the apoptotic pathway inresponse to external force. The physiologic roles of
ADRB2 and TIEG have been proposed by characterizing genetically modified mouse models (Elefteriou et al.,2005; Subramaniam et al., 2005). The Adrb2-deficient
mice showed that the sympathetic nervous system favors
bone resorption by increasing expression in osteoblastprogenitor cells of the osteoclast differentiation factor,
receptor activator of NF-κB ligand (Rankl). On the other
hand, the analysis of Tieg-deficient osteoblasts revealed
that TIEG expression is critical for both osteoblastmediated mineralization and osteoblast support ofosteoclast differentiation. Thus, ADRB2 and TIEG may

K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

be crucial molecules in HPLF for regulation of bonehomeostasis.
Inhibition assays using Y27632 revealed that static
HPLF failed to develop stress fibers as visualized by
F-actin staining. Y27632 inhibits two isoforms of a serine/threonine kinase, termed ROCK-I and II, although
Y272632 is not completely specific for these kinases
it has a strong negative impact on ROCK-induced
cellular functions (Davies, Reddy, & Caivano, 2000;
Rottner, Hall, & Small, 1999). It has been recognizedthat inactivation of ROCKs (or ROCK-I) causes a lossof stress fibers and focal adhesion (Riento & Ridley,
2003; Yoneda, Multhaupt, & Couchman, 2005), and
Rho/ROCK-dependent cytoskeletal reorganization has
been reported in various cells and tissues. Howeverinactivation of the Rho/ROCK axis has different effects
on cell behavior, such as differentiation, apoptosis or
tumorigenesis (Hakuma et al., 2005; McBeath, Pirone,
Nelson, Bhadriraju, & Chen, 2004; Svoboda, Moessner,
Field, & Acevedo, 2004), depending on the cell context
and cell type analyzed. Thus, Rho/ROCK might to be
included in several groups of signaling molecules that
may regulate the variety of downstream signals controlling the phenotypic changes in the cells, which need
to be further examined in HPLF. It has been reported
that Y27632 potently stimulated bone morphogenetic
protein (BMP)-2/4 expression, osteoblast differentiation, and bone nodule formation (Harmey, Stenbeck,
Nobes, Lax, & Grigoriadis, 2004), suggesting a possible
mechanism for bone metabolism involving periodontal
ligament cells and osteocytes regulated by Rho/ROCK.
Y27632 selectively altered some CT gene expression
such as ACY1, ATF1, GOSR1 however Y27632 exposure did not significantly alter the expression of other CT
genes. It has been reported that the mammalian homologue of Drosophila Diaphanous protein 1 (Dia1) is alsoa crucial downstream target of Rho, that regulates actin
polymerization and microtubule organization (Ishizaki
et al., 2001; Palazzo, Cook, Alberts, & Gundersen,
2001). Among multiple Rho targets, it has been
described that the combined action of ROCK and Dia1
are sufficient to substitute for active Rho in the process of
mechanotransduction (Watanabe, Kato, Fujita, Ishizaki,& Narumiya, 1999), moreover the growth of focal adhesions can be achieved when mechanical force is appliedeven if ROCK activity is suppressed by Y27632 (Geiger& Bershadsky, 2001; Riveline et al., 2001). Thus, there
is a possibility that the Rho–ROCK pathway can bebypassed if tension is applied externally, and that Dia1
could partially compensate for the cellular response tomechanotransduction in HPLF when ROCK is inactive(Fig. 6). Fos induced-Rho/ROCK-independent pathways

919

Fig. 6. Schematic representation of CT genes expression. Cyclic
tension-induced focal adhesion, stress fibers and CT genes expression
are mediated by activation of Rho-Dia1 or Rho–ROCK that is inhibited by Y27632. Early responsive gene, FOS expression is increased
when cyclic tension is applied even if ROCK activity is suppressedby Y27632. Y27632 does not alter TP53BP2, ADRB2, and BCL2
(ROCK-independent) genes expression as well, while Y27632 effectively recover ACY1, ATF1, and GOSR1 (ROCK-dependent) genes
expression to the basal level. Dia1 might partially compensate mechanotransduction in HPLF when ROCK is inactive. Rho, Ras-homologof small GTPase; ROCK, Rho-associated kinases; Dia1, mammalian
homologue of Drosophila Diaphanous protein 1. Abbreviations of CT
genes can be found in Supplementary-4.

may tightly control HPLF survival through regulationof BCL2, TP53BP2 or IER3, and bone remodeling by
ADRB2 or TIEG. To clarify these signaling pathways
regulating CT gene expression, we need to further examine the kinetics of mRNA expression and phenotypes ofROCK- or Dia1-knockdown HPLF in the next study.
In conclusion, oligonucleotide array analysis revealed
that 122 CT genes had significantly altered levels of
expression in tension-loaded HPLF, and that typicalkinetic patterns and functional classifications existed for
the CT genes. By combining the K-means cluster and
IPA, we found temporal and spatial alterations of CT
gene expression, and the presence of several signaling
pathways likely responsible for controlling the responsein HPLF mechanotransduction. Some of these pathways
were not dependent on Rho–ROCK activity, and thus theoverall response may be compensated by other signaling
pathways. The complex regulation of mechanotransduction in HPLF needs to be further analyzed to allow
application of these findings to control the regenerationof periodontal ligament.
Acknowledgements
This study was supported by a Grant-in-Aid for
Scientific Research B (No. 14370710 to ST), a Grant-

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K. Yamashiro et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 910–921

in-Aid for Scientific Research A (Nos. 16209056 and18209057), a Grant-in-Aid for Scientific Research B(No. 17390562 to FN), a Grant-in-Aid for Scientific
Research C (No. 17591991 to HA), and a Grant-inAid for Scientific Research A1 (No. 16209063 to YA)
from the Japan Society for the Promotion of Science,
Kobayashi Magobe Memorial Foundation, Ryobi Teien
Foundation, and Inamori Foundation (FM), and Suzuken
Memorial Foundation (ST). We would like to thank Dr.
Charles F. Shuler, Director of Center for Craniofacial
Molecular Biology, University of Southern California,
for reviewing the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.biocel.2007.01.015.
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doi: 10.1111/j.1600-0714.2007.00515.x

J Oral Pathol Med (2007) 36: 223–8ª Blackwell Munksgaard 2007 Æ All rights reserved
www.blackwellmunksgaard.com/jopm

Microarray analysis of IL-1b-stimulated chemokine genes
in synovial fibroblasts from human TMJ
Naomi Ogura1,2, Miwa Akutsu1, Makiko Tobe1,2, Hiroyuki Sakamaki1,2, Yoshimitsu Abiko2,3,
Toshirou Kondoh1,21Department of Maxillofacial Surgery, Nihon University School of Dentistry at Matsudo, Chiba; 2Research Institute of Oral Science,
Nihon University School of Dentistry at Matsudo, Chiba; 3Department of Biochemistry and Molecular Biology, Nihon University
School of Dentistry at Matsudo, Chiba, JapanBACKGROUND: Interleukin (IL)-1b is thought to play a
key role in several pathologic conditions of the temporomandibular joint (TMJ). Gene expression profile ofsynovial fibroblasts stimulated with IL-1b was studied byoligonucleotide microarray analysis to elucidate candidate genes associated with intracapsular pathologic conditions of TMJ.
METHODS: RNA was isolated from synovial fibroblasts
from five patients after IL-1b treatment. Gene expression
profiling was performed with a GeneChip. Changes
in gene expression were determined by comparing
IL-1b-treated cells with untreated cells.
RESULTS: A total of 121 genes showed a greater than
threefold difference in average intensity between untreated and IL-1b-treated synovial fibroblasts in fiveexperiments. Five chemokines were among the 10 mostupregulated genes, and the most upregulated gene was
CCL20. The 121 IL-1b-responsive genes included 12
chemokines whose mRNA levels were confirmed by realtime PCR.
CONCLUSION: These data should provided useful
information about the pathologic conditions of TMJ,
especially in support of diagnosis and therapeutic
approaches to TMJ.
J Oral Pathol Med (2007) 36: 223–8
Keywords: chemokine; interleukin-1b; oligonucleotide microarray; synovial fibroblasts; temporomandibular joint

Introduction
Synovitis, which often accompanies intracapsular pathologic conditions such as disk displacement (DD)/
Correspondence: Toshirou Kondoh, Department of Maxillofacial
Surgery, Nihon University School of Dentistry at Matsudo, 2-870-1,
Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan. Tel: +81 47 360
9394, Fax: +81 47 360 9394, E-mail: kondo.toshiro@nihon-u.ac.jp
Accepted for publication November 21, 2006

internal derangement (ID) and osteoarthritis (OA) of
the temporomandibular joint (TMJ), is characterized bychronic inﬂammatory changes, including hyperplasia of
the synovial lining (1, 2), growth of small new bloodvessels (1), and inﬁltration of inﬂammatory cells (1, 3–5). In addition, synovial cells produce a number ofputative mediators of inﬂammation (6, 7).
Interleukin (IL)-1b is a proinﬂammatory cytokine
that aﬀects cell proliferation (8), inﬂammatory responses(9), and matrix remodeling (10). Elevated expression of
IL-1b in the joint is known to result in activation of
inﬂammatory and degradative pathways in synovial
cells, which in turn contribute to the progression ofrheumatoid arthritis (RA; 8, 11). Under pathologic
conditions of the TMJ, levels of IL-1b are elevated insynovial ﬂuids obtained from patients with OA and RA(12). Studies have also indicated that IL-1b in synovial
ﬂuid is associated with pain in the TMJ (13, 14).
However, the involvement of synovial cells underpathologic conditions in the TMJ is poorly understood,
as synovial cells have been diﬃcult to cultivate inquantities suﬃcient for study.
Our group has isolated synovial cells from the humandiseased TMJ and examined how regulation of IL-1baﬀects the underlying inﬂammatory status (15–17). We
hypothesized that identiﬁcation of IL-1b-responsive
genes in synovial ﬁbroblasts could help develop models
for the inﬂammatory condition of the TMJ. However,obtaining an overview of the molecular and cellularresponses of synovial ﬁbroblasts to IL-1b has beendiﬃcult, in part because classic approaches for studying
inﬂammatory factors have focused on identiﬁcation andanalysis of speciﬁc postulated factors on an individual
basis. DNA microarray techniques have recently beendeveloped to facilitate comprehensive expression analysis of large numbers of deﬁned genes (18–20). Microarray analysis is an eﬃcient method for exploring thefunctions of uncharacterized genes, as the expression
pattern of uncharacterized genes can be comparedwith the expression pattern of characterized genes.

IL-1b-induced genes in synovial fibroblasts
Ogura et al.

224

Microarray analysis can also be used to search fordiﬀerentially expressed genes, which may include diagnostic and therapeutic markers.
In order to understand how IL-1b can induced theintracapsular pathologic condition of the TMJ, we
examined the gene expression proﬁles of non-induced
and IL-1b-induced synovial ﬁbroblasts from the human
TMJ using an oligonucleotide microarray approach.

Materials and methods
Cell culture
Human synovial tissue was obtained from ﬁve patientswith ID who underwent arthroscopy of the TMJ (threefemales and two males; age range: 17–27 years). Patientsgave complete informed consent for the surgery and forthe use of their tissue in research. Isolation and primarycultures of synovial ﬁbroblasts were performed according to the guidelines established by the Institutional
Review Board of Nihon University School of Dentistry
at Matsudo (EC03-003).
Synovial ﬁbroblasts from the TMJ were prepared aspreviously described (15). For the experiments, we usedsynovial ﬁbroblasts from the sixth to eighth passages.
Total RNA extraction
Synovial ﬁbroblasts were incubated with or without
1 unit/ml IL-1b for 4 h. Total cellular RNA fromsynovial ﬁbroblasts (TMJ 1–5) was extracted using
RNeasy kits (Qiagen, Valencia, CA, USA), and wasthen stored at )80C until used.
GeneChip expression analysis
For gene expression proﬁling, we used the Aﬀymetrix
GeneChip Human Genome Focus Array (Aﬀymetrix,
Santa Clara, CA, USA) according to Aﬀymetrix protocols. Brieﬂy, double-stranded cDNA was generated
from 7 lg of total RNA using a T7-oligo (dT) primer
and the Superscript Choice System kit (Invitrogen,
Carlsbad, CA, USA). cDNA was then puriﬁed with a
GeneChip Sample Clean-up Module (Aﬀymetrix). Biotin-labeled cRNA was synthesized by in vitro transcription using the BioArray high-yield RNA transcript
labeling system (ENZO, Farmingdale, NY, USA).
cRNA was then puriﬁed using a GeneChip Sample
Clean-up Module. Biotin-labeled cRNA was added to
the fragmentation buﬀer (Aﬀymetrix), and was heatedfor 35 min at 95C. Fragmented cRNA (10 lg) was
hybridized to the Human Genome Focus Array (8793
genes) for 16 h at 45C. Arrays were subjected towashing and staining with R-phycoerythrin streptavidin(SAPE, Molecular Probes, Eugene, OR, USA) using
GeneChip Fluidics Station 400 (Aﬀymetrix).
In order to amplify the signals, the arrays were further
stained with goat biotinylated antistreptavidin antibody(Vector Laboratories, Burlingame, CA, USA), followedby SAPE. After scanning (scanner from Aﬀymetrix), theoligonucleotide hybridization data were exported forgene expression value analysis using AFFYMETRIX MICROARRAY SUITE (version 5.0). The hybridization intensity
for each gene was calculated with a probe set containing
J Oral Pathol Med

24 speciﬁc probes for perfect matches and mismatches.
The expression of each gene was also categorized aspresent’, absent’, or marginal’. For quality control,cRNA samples were hybridized to the test microarraychip (Test 3; Aﬀymetrix) in order to ensure equalhybridization to 5¢- and 3¢-oligonucleotides of the genes
chosen for standardization (GAPDH and b-actin)
before hybridization with Focus Array.
Data analysis
Raw data from 10 GeneChips were loaded into GENESPRING software (version 6.2; Agilent Technologies,
Waldbronn, Germany). Data were normalized using
the median raw data from each array as a reference.
Changes in gene expression were determined by comparing controls (average normalized intensity of untreated cells) with IL-1b treatment (average normalized
intensity of IL-1b-treated cells).
Real-time PCR
cDNA was synthesized using a GeneAmp RNA PCR kit(Perkin-Elmer, Norwalk, CT, USA). Real-time PCR
was performed using a DyNAmo SYBR Green qPCR
kit (Finnzymes, Espoo, Finland). The PCR mixture
contained 20 pmol of forward and reverse primers and2 ll of cDNA. Ampliﬁcation was performed using a
DNA Engine Opticon 1 (MJ Research, San Francisco,
CA, USA), with pre-heating at 95C for 10 min,followed by 40 cycles of 94C for 15 s, 57C for 30 s,
and 72C for 30 s. Amplicons were detected directly bymeasuring the increase in ﬂuorescence caused by thebinding of SYBR Green I dye to gene-speciﬁc, ampliﬁed, double-stranded DNA (SYBR ﬂuorescence systems
in real-time PCR can be used to semiquantitatively
analyze mRNA expression with on-line monitoring).
Following the completion of PCR ampliﬁcation, thetemperature was raised from the annealing temperature
to 95C for melting curve analysis.
The initial template concentration was derived fromthe cycle number at which the ﬂuorescent signal crosseda threshold (CT) in the exponential phase of PCR. The
number of transcripts was determined based on thethreshold cycles of chemokines and GAPDH. DCT (CTchemokine minus CT-GAPDH) indicates the relative
amount of chemokine transcripts. DDCT (DCT-treatedminus DCT-control) represents the relative n-value
compared with the control. The quantity 2)n represents
the diﬀerence in chemokine expression between IL-1btreated cells and controls. Chemokine primer sequences
are given in Table 1.

Results
The expression of 8793 genes on the Human Genome
Focus Array was compared between controls (averagevalues of ﬁve non-treated cells from ﬁve patients) andIL-1b-treated cells (average values of IL-1b-treated cells
from ﬁve patients). A total of 121 genes showed agreater than threefold diﬀerence in intensity between
control and IL-1b-treated synovial ﬁbroblasts; 111 wereupregulated and 10 were downregulated.

IL-1b-induced genes in synovial fibroblasts
Ogura et al.

225

Table 1 Primers for chemokine
Chemokine

Primersbp

CCL20 (MIP-3)

F: 5¢-GCA AGC AAC TTT GAC TGC TG-3¢
R: 5¢-CAA GTC CAG TGA GGC ACA AA-3¢
F: 5¢-TAA ATG ACA GGG TGG GGA AC-3¢
R: 5¢-GCA TTA TGC CCT ACA AGC AA-3¢
F: 5¢-CCT GGA CAA GAA AAC CCA AA-3¢
R: 5¢-TTC AAA ACC CAC CAA AAT CC-3¢
F: 5¢-CCC TGC CTT ACA GGA ACA GAA-3¢
R: 5¢-TCC CTG CCG TCA CAT TGA TCT-3¢
F: 5¢-TGC AAG CCA ATT TTG TCC ACG TGT TG-3¢
R: 5¢-GCA GCT GAT TTG GTG ACC ATC ATT GG-3¢
F: 5¢-TGC AGG GAA TTC ACC CCA AG-3¢
R: 5¢-CAG GGC CTC CTT CAG GAA CA-3¢
F: 5¢-ATC ACT TCC AAG CTG GCC GTG GCT-3¢
R: 5¢-TCT CAG CCC TCT TCA AAA ACT TCT C-3¢
F: 5¢-TGC AAA ATC CTG GTG ATG TG-3¢
R: 5¢-AGG AGC ACT GAT TGC CAA AG-3¢
F: 5¢-GAG TGG GTC CAA TGC ACT TT-3¢
R: 5¢-CAC AGA CGT TGG TGA TGA GG-3¢
F: 5¢-CAA TGA TCT GTG CTC TGC AA-3¢
R: 5¢-CCA ACA TGA CAC ACA GGA AAA-3¢
F: 5¢-TAC ACC AGT GGC AAG TGC TC-3¢
R: 5¢-GAA GCC TCC CAA GCT AGG AC-3¢
F: 5¢-CCA ATT CTC AAA CTG AAG CTC GCA-C3¢
R: 5¢-GTT AGC TGC CAG ATT CTT GGG TTG TG-3¢
F: 5¢-ATC ACC ATC TTC CAG GAG-3¢
R: 5¢-ATC GAC TGT GGT CAT GAG-3¢

342

CXCL3 (GRO-c)
CCL7 (MCP-3)
CXCL2 (GRO-b)
CXC10 (IP-10)
CXCL1 (GRO-a)
CXCL8 (IL-8)
CCL8 (MCP-2)
CX3CL1 (fractalkine)CXCL6 (GCP-2)
CCL5 (RANTES)
CCL2 (MCP-1)
GAPDH

225
245
403
302
229
289
166
241
218
199
372
315

CCL, CC motif chemokine ligand; CXCL, CXC motif chemokine ligand; CX3CL, CX3C motif chemokin ligand; MIP, macrophage inﬂammatory
protein; GRO, growth-regulated oncogene; MCP, monocyte chemoattractant protein; IP, interferon-inducible protein; IL, interleukin; GDP,
granulocyte chemotactic protein; RNATES, regulated upon activation, normal T expressed and secreted; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.

Table 2 Upregulated genes by treatment with IL-lb

Ranka

Gene

GenBank

Fold changeb(IL-lb/control)

Gene title Aﬀy

1
2
3
4
5
6
7
8
9
10

CCL20
CXCL3
BCL2A1
PTGS2
CXCL2
IL-8
CSF3
CCL7
IL-1b
IL-6

NM_004591
NM_002090
NM_004049
NM_000963
M57731
AF043337
NM_000759
NM_006273
M15330
NM_000600

413.6
60.8
41.0
38.9
36.3
35.6
29.4
21.0
20.5
19.9

Chemokine (C–C motif) ligand 20
Chemokine (C–X–C motif) ligand 3
BCL2-related protein A1
Prostaglandin-endoperoxide synthase 2
Chemokine (C–X–C motif) ligand 2
Interleukin 8
Colony-stimulating factor 3 (granulocyte)
Chemokine (C–C motif) ligand 7
Interleukin 1, b
Interleukin 6aRanking of upregulated gene by IL-1b.
Average normalized intensitiy of IL-lb-sitmulated cell in TMJ 1–5/average normalized intensity of control cell in TMJ 1–5.

b

Table 2 lists the top 10 upregulated genes. The genemost strongly upregulated by IL-1b (rank 1) was CCL20(MIP-3a), a member of the chemokine superfamily. In
fact, ﬁve chemokines were among the top 10 upregulated
genes; CXCL3 (GRO-c; rank 2), CXCL2 (GRO-b; rank5), IL-8 (CXCL8; rank 6), and CCL7 (MCP-3; rank 8).
The remaining ﬁve genes in the top 10 were BCL2A1(BCL-related protein A1), PTGS2 (prostaglandin-endoperoxide synthetase 2), CSF3 (granulocyte colony-stimulating factor; G-CSF), IL-1B (IL-1b), and IL-6.
The 121 IL-1b-responsive genes, which showed agreater than threefold diﬀerence between control andIL-1b-treated synovial ﬁbroblasts, were categorizedbased on the ontology of their molecular function(Fig. 1). The category with the most genes was signaltransduction (34 genes; 32 upregulated and two downregulated), followed by inﬂammatory response (20genes; 20 upregulated), proliferation (15 genes; 13
upregulated and two downregulated), and chemotaxis(12 genes; 12 upregulated). Inﬂammatory response (20of 139 genes, 14.4%) and chemotaxis (12 of 88 genes,
13.6%) accounted for the largest percentage of IL-1bresponsive genes among those identiﬁed on HG Focus
Array. All of the 12 chemotaxis genes were also
categorized as inﬂammatory response genes, such as
chemokine superfamily members.
J Oral Pathol Med

IL-1b-induced genes in synovial fibroblasts
Ogura et al.

226

Cell cycle (9/308, 2.9%) 2

7

Cellular defense (4/62, 6.5%)

0Chemotaxis (12/88, 13.6%)

0Induction of apoptosis (3/92, 3.3%)

0

Inflammatory response (20/139, 14.4%)

0

Phosphorylation (8/378, 2.1%)

0

4
12
3
20
8

Proliferation (15/370, 4.1%) 2

13

Signal transduction (34/934, 3.6%) 2

32

Figure 1 Ontology for IL-1b-responsive genes. The categories of each gene are indicated (number of IL-1b-responsive genes/number of genes
identiﬁed on HG Focus Array, percentage of IL-1b-responsive genes).

Table 3

Levels of IL-lb-responsive chemokines mRNA in synovial ﬁbroblasts from TMJ
Fold (IL-Ib/control)

Chemokine

GenBank

GeneChipReal-time PCR

Chemoattract target cells

CCL20 (MIP-3a)
CXCL3(GRO-c)
CCL7 (MCP-3)
CXCL2(GRO-b)
CXC10 (IP-10)
CXCL1 (GRO-a)
CXCL-8(IL-8)
CCL8(MCP-2)
CX3CL1 (fractalkine)

NM_004591
NM_002090
NM_006273
NM_002089
NM_001565
NM_001511
NM_000584
NM_005623
NM_002996

545.2
61.6
47.0
34.5
13.8
11.7
11.3
9.9
9.5

224.6
158.3
86.4
83.7
87.5
72.4
77.4
50.8
8.9

CXCL6(GCP-2)
CCLS(RANTES)
CCL2(MCP-1)

NM_002993
NM_002985
NM_002982

6.4
4.0
3.5

19.3
8.3
7.2

Memory T cell, immature dendritic cell
Neutrophil, endothelial cell
Monocyte, Th1 cell, eosinophil basophil, NK cell, dendritic cell
Neutrophil, melanoma cell
Monocyte, Th1 cell, NK cell
Neutrophil, lymphocyte, monocyte
Neutrophil, CD8+ T cell, NK cells, monocyte
CD4+ T cell, CD8+ T cell, monocyte, eosinophil, basophil, NK cell
NK cell, intraepithelial lymphocyte, CD8+ T cell,CD4+ T cell, monocytes
Neutrophil
Eosinophil, T cell, monocyte, basophil
Monocyte, T cell, basophil

CCL, CC motif chemokine ligand; CXC, CXC motif chemokine ligand; CX3CL, CX3C motif chemokin ligand; MIP, macrophage inﬂammatory
protein; GRO, growth-related gene product; MCP, monocyte chemoattractant protein; IP, interferon-inducible protein; IL, interleukin; GDP,
granulocyte chemotactic protein; RNATES, regulated upon activation, normal T expressed and secreted.

Discussion
Changes in the expression and production of inﬂammatory factors have an impact on both homeostasis and thepathologic condition of the TMJ. We hypothesized that
identiﬁcation of IL-1b-responsive genes in synovial
J Oral Pathol Med

100

CCL20

IL-8

CXCL1 CCL2

CXCL2
CXCL3
CXCL6
CCL7
IL-1β (Normalized intensity)

We focused on genes that encode chemokine superfamily members, as this gene family exhibits the mostsigniﬁcant responses to IL-1b (Table 2 and Fig. 1). The
12 chemokines included ﬁve CC motif chemokines, six
CXC motif chemokines, and one CX3C motif chemokines (Table 3). Figure 2 shows scatter plots of the 121
IL-1b-responsive genes and the 12 indicated chemokines. CCL20, CCL2, IL-8, CXCL1, CXCL2, andCXCL3 exhibited high expression in IL-1b-treated cells.
Table 3 shows the IL-1b-induced fold-change using
microarray and real-time PCR analysis in the TMJ 5
sample. Although the fold-change between the control
and IL-1b-treated synovial ﬁbroblasts determined byreal-time PCR diﬀered from that found on microarray,
the rank order of the IL-1b response was similar
between microarray and real-time PCR (Table 3).

10
CX3CL1CCL8
CCL51CXCL10

0.1

0.01
0.01

0.1
1
10
Control (normalized intensity)

100

Figure 2 Chemokines in scatter plots of IL-1b-responsive genes.
Normalized intensity was calculated from ﬁve experiments using
samples TMJ 1–5.

IL-1b-induced genes in synovial fibroblasts
Ogura et al.

ﬁbroblasts could help develop models for the inﬂammatory condition of the TMJ. Cell culture systems
provide a well-established means for initial identiﬁcationof cytokine-responsive genes. Of the cells from patientswith intracapsular pathology of the TMJ, we decided toanalyze the cells that produced the lowest levels ofcytokines under untreated conditions, as we were unable
to obtain synovial cells from healthy individuals.
We performed oligonucleotide microarray analysis inorder to examine the consistency of gene regulation insynovial ﬁbroblasts treated with IL-1b. Among the 8793
genes tested, a total of 121 exhibited a greater than
threefold change in intensity between control and IL-1btreated synovial ﬁbroblasts, and these genes werecategorized based on their molecular functions. Manyof the upregulated genes can be categorized as signaltransduction’ or inﬂammatory response’ genes. It has
previously been suggested that IL-1b contributes to the
production of inﬂammatory factors by modulating theexpression of signal transduction.
Numerous chemokine superfamily members, whichwere categorized inﬂammatory response and chemotaxis
genes, were also among the top 10 IL-1b-responsive
genes identiﬁed in this study. Chemokines are considered key players in the diapedesis of leukocytes from thevasculature into tissues in inﬂammatory diseases (21–23). Inﬂammatory arthropathies are characterized histologically by inﬁltration of inﬂammatory cells andenlargement of the synovial lining layer (24, 25).
Accumulation of neutrophils, activated T cells, andmacrophages in inﬂamed synovial tissues may lead tosigniﬁcant structural damage to joints with RA (11, 24,
25). Inﬂammatory cells have also been detected insynovial tissue and in ﬂuid from patients with intracapsular pathologies of the TMJ (1, 3, 5). The mechanisms
leading to cellular inﬁltration of the synovium and joint
degeneration have been elucidated to a degree by
studying the release of degradative enzymes (26),various products of oxidative metabolism (27), andinﬂammatory cytokines (24). The following sequence of
events is consistent with these ﬁndings: (i) chemokines
produced by synovial ﬁbroblasts stimulate chemotaxisof neutrophils, macrophages, and T lymphocytes; (ii)these inﬂammatory cells produce inﬂammatory cytokines such as IL-1b, matrix degradative enzymes, andvarious products of oxidative metabolism; (iii) the
enzymes and oxidative metabolites cause degradationof the extracellular matrix; (iv) the inﬂammatory cytokines stimulate synovial ﬁbroblasts to produce morechemokines. Furthermore, the subset of CXC-type
chemokines that contain the sequence Glu-Leu-Arg(the ELR’ motif), CXCL1, 2, 3, 6, 8, are also thought topromote angiogenesis (28). Thus, induction of the ELRCXCs may lead to both recruitment of inﬂammatory
cells and new small vessels in synovial tissues. Althoughchemotaxis is a necessary function of homeostasis,
inappropriate inﬁltration of inﬂammatory cells may
cause joint degeneration.
The other genes among the top 10 responsive genes
were BCL2A1 (Bﬂ-1/A1 protein), PTGS2 (COX2),
CSF3 (G-CSF), IL-1B, and IL-6. Many reports havedemonstrated that prostaglandin E2 synthesized by
COX2, G-CSF, IL-1b, and IL-6 is increased insynovial tissues and ﬂuids with severe inﬂammation,such as in RA or OA (29–31). Bﬂ-1/A1 protein is amember of the Bcl-2 family, which also includes Bcl-2
and Bcl-x (L), and suppresses apoptosis (32). Thisfactor may be associated with hyperplasia of thesynovial lining.
The development and application of genome-scaletechnologies for studying the IL-1b response may help
to understand inﬂammation and develop new treatments. This study looked at changes in the gene
expression of chemokines in response to IL-1b insynovial ﬁbroblasts from the human TMJ using microarray. We also compared the gene expression levels
detected by microarray and real-time PCR methods.
Although the fold-change between control and IL-1btreated synovial ﬁbroblasts measured by real-time PCR
diﬀered slightly from that on microarray analysis, therank order of IL-1b-responsive genes was similar, thus
suggesting that gene chip analysis is a valid andpowerful tool for analyzing the expression of a large
number of molecules.
We used microarray analysis to identify diﬀerentiallyexpressed genes in order to characterize synovial ﬁbroblasts. As expected, synovial ﬁbroblasts express vimentin, prolyl 4-hydroxylase, and type I collagen, which areﬁbroblast markers. In contrast, the expression of HLAclass II antigens were not detected (data not shown). We
observed no signiﬁcant diﬀerences in the gene expression
levels of cell markers between control and IL-1b-treatedcells. These data were consistent with the notion thatsynovial ﬁbroblasts are ﬁbroblasts-like cells with no rolein antigen presentation.
In conclusion, our comparative gene expression proﬁle analysis has revealed interesting features of synovial
ﬁbroblasts from human TMJ. Upon IL-1b stimulation,
synovial ﬁbroblasts expressed high levels of several
chemokines, suggested that IL-1b promotes the recruitment of leukocytes to the inﬂammation sites in TMJ
synovium. These ﬁndings demonstrated that synvoial
ﬁbroblasts in TMJ should be considered as important as
its surrounding cells or tissue in the initiation or
progression of inﬂammatory TMJ. In addition, we alsosuggest that the present data will be useful for theidentiﬁcation of candidates for genes with key roles in
the initiation and progression of pathologic intracapsular conditions of the TMJ.

227

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Acknowledgements
We gratefully acknowledge the assistance of Asayo Imaoka regarding
GeneChip technology. This study was supported by a Grant-in-Aid for
Scientiﬁc Research (c) (17592111) from the Japan Society for the
Promotion of Science, and by a Grant for Research at the Frontier of
Science from the Ministry of Education, Science, Sports and Culture(2001).

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Effects of reactive oxygen species (ROS) on antioxidant
system and osteoblastic differentiation in MC3T3-E1
cells
Masato Arai a; Yasuko Shibata a; Kamolparn Pugdee a; Yoshimitsu Abiko a;
Yorimasa Ogata aaDepartment of Periodontology, Nihon University School of Dentistry at Matsudo,
Chiba, Japan

Online Publication Date: 01 January 2007
To cite this Article: Arai, Masato, Shibata, Yasuko, Pugdee, Kamolparn, Abiko,
Yoshimitsu and Ogata, Yorimasa (2007) 'Effects of reactive oxygen species (ROS)
on antioxidant system and osteoblastic differentiation in MC3T3-E1 cells', IUBMBLife, 59:1, 27 - 33
To link to this article: DOI: 10.1080/15216540601156188
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IUBMB

Life, 59(1): 27 – 33, January 2007

Research Communication
Effects of Reactive Oxygen Species (ROS) on Antioxidant System
and Osteoblastic Differentiation in MC3T3-E1 Cells
Masato Arai, Yasuko Shibata, Kamolparn Pugdee, Yoshimitsu Abiko and Yorimasa Ogata
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, Japan

Summary
Oxidative stress regulates cellular functions in multiple pathological conditions, including bone formation by osteoblastic cells.
However, little is known about the cellular mechanisms responsible
for the eﬀects of oxidative stress on osteoblast functions insenescence. To clarify the inhibitory eﬀects of oxidative stress on
osteoblastic mineralization, we examined the relationship between
the antioxidant system and bone formation in MC3T3-E1 cells.
After a single exposure to H2O2 within range of a non-toxic
concentration for cells, the mineralization level was diminished half.
Under the same conditions, gene expression of the transcription
factor Nrf2, which regulates antioxidant enzymes, was upregulated. In addition, gene expression for the osteogenic markers
Runx2, ALP, and BSP was lower than that in non-treated cells,
whereas expression of the osteocalcin gene was up-regulated
following H2O2 exposure. These results suggest that reduced
mineralization by MC3T3-E1 cells after H2O2 exposure is theresult of an up-regulated antioxidant system and altered osteogenic
gene expression.
IUBMB Life, 59: 27–33, 2007
Keywords

Osteoblastic diﬀerentiation; reactive oxygen species(ROS); senescence; antioxidant; Nrf2.

INTRODUCTION
Reactive oxygen species (ROS), such as hydrogen peroxide(H2O2), hydroxyl radicals, and superoxide, are generated
under various physiological conditions and believed to contribute to the etiology of atherosclerosis, cerebellar ischemia,

Received 27 October 2006; accepted 6 December 2006
Address correspondence to: Yorimasa Ogata, Department of
Periodontology, Nihon University School of Dentistry at Matsudo,
2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan. Tel: þ81
47 360 9362. Fax: þ81 47 360 9362.
E-mail: ogata.yorimasa@nihon-u.ac.jp
ISSN 1521-6543 print/ISSN 1521-6551 online Ó 2007 IUBMBDOI: 10.1080/15216540601156188

and the process of aging (1 – 5). Further, oxidative stressinduced by ROS has been implicated in the induction ofcertain types of cell injury (6, 7). However, a recently
proposed hypothesis states that ROS plays an importantrole in the regulation of cell proliferation and metabolism(8 – 10).
In bone tissues, both formation and maintenance areregulated by bone-forming osteoblasts and bone-resorbing
osteoclasts (11 – 13), while an imbalance between those twocell types leads to the pathogenesis and etiology of certainbone metabolic diseases, including osteoporosis and osteopetrosis (14). Recent evidence has shown that ROS andantioxidant enzymes might be involved in the pathogenesisof bone loss such as osteoporosis (15, 16). In addition,
Ozgocmen et al. (17) reported that antioxidant enzyme
activities that serve as oxidative stress markers, such as
superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and lipid peroxidation end-product malondialdehyde(MDA), might be important indicators of bone loss inpostmenopausal women. In experimental studies, the eﬀects
of ROS on genes involved in osteoblast proliferation anddiﬀerentiation remain unclear, in part because of the sometimes conﬂicting results observed with diﬀerent stages ofcultured osteoblastic cells and various oxygen tensions. Also,
the process of osteoblast diﬀerentiation leading to bonemineralization is a poorly understood series of temporally
and spatially coordinated events that occur within weeks ormonths. A signiﬁcant end-point in this process is creation of amineralized matrix that consists mainly of collagen, noncollagenous proteins, and hydroxyapatite. Extracellular aging,which is caused by accumulation of molecular damage byoxidation, glycation, and crosslinking of long-living extracellular proteins, is a major cause of several important
human aging pathologies in bone tissue. We have shown thatoxidized ﬁbronectin (FN) induces the reduction of nodule
formation with decreases in alkaline phosphatase (ALP)
activity and levels of secretion of type I collagen in rat
calvarial osteoblasts in vitro (18, 19). However, there is scantDownloaded By: [Nihon University] At: 01:34 16 April 2008

28

ARAI ET AL.

information regarding antioxidant systems in osteoblastic
diﬀerentiation.
The sensitive cellular response to mild oxidative stressinduces activation of several antioxidant enzymes that play animportant role in the protection of cells against ROS andfacilitate the prevention of oxidative stress-induced metabolism (20). In addition, the transcription factor nuclear factor
E2 p45-related factor 2 (Nrf2) is one of the critical transcription factors involved in the regulation of antioxidant
enzymes (21, 22). Nrf2 forms heterodimers with small Maf
proteins for selective recognition of the antioxidant-responsive
element (ARE) or electrophile-responsive element on target
genes (21 – 23).
Recently, a study by Hinoi et al. (24) using MC3T3-E1
cells stably transfected with an nrf2 expression vector
indicated that Nrf2 negatively regulates osteoblast diﬀerentiation through inhibition of runt-related transcription
factor 2 (Runx2)-dependent transcriptional activity. In thepresent study, we attempted to reassess the eﬀects ofantioxidant systems on osteoblast diﬀerentiation, and also
determined gene expressions for antioxidant-related andosteogenic marker molecules using hydrogen peroxide(H2O2)-treated MC3T3-E1 cells. To determine the eﬀects
of oxidative stress on MC3T3-E1 cells, we treated the cells
directly with H2O2, as we have observed that H2O2 stressinhibits diﬀerentiation of MC3T3-E1 cells. Using identicalconditions, we found that a single exposure to H2O2 led to
an up-regulated antioxidant system and down-regulated
the gene expression of osteogenic marker molecules in
MC3T3-E1 cells.

MATERIALS AND METHODS
Cell Culture and H2O2 Treatment
MC3T3-E1 cells were purchased from Cell Bank (Riken
BioResource Center, Ibaraki, Japan) and cultured in alphaminimal essential medium (a-MEM, plus L-glutamine, ribonuculeosides, and deoxyribonucleosides, Invitrogen Co. CA)
containing 10% fetal bovine serum (FBS) supplemented with
antibiotics (50 mg/ml gentamycin sulfate, 0.3 mg/ml amphotericin B, 100 units/ml penicillin G potassium) at 378C in a CO2
incubator (5% CO2/95% air). MC3T3-E1 cells were subcultured at approximately 80% conﬂuence in every 3 days in
75-cm2 ﬂasks.
To investigate the eﬀects of ROS as an in vitro cellularsenescence, we prepared the H2O2-treated MC3T3-E1 cellsby the method of Chen et al. (25) with slight modiﬁcation. MC3T3-E1 cells were plated at a high density(16105 cells/ml, 0.5 ml/well) to provide near conﬂuence ina 24 well plate. After 2 days, the cells were treated with
a-MEM containing H2O2 and cultured for 3 h. Subsequently,
they were washed with fresh a-MEM to omit H2O2. To
observe osteoblastic diﬀerentiation, a-MEM containing 10%
FBS, antibiotics, 50 mg/ml of ascorbic acid, and 5 mM ofb-glycerophosphate (b-GP) were used. The culture medium
was changed to fresh every 3 days throughout the 50 day
experimental period. The viability of MC3T3-E1 cells after
H2O2 treatment in each concentration was assessed using
Alamar Blue assay (Biosource International, CA). Alamar
Blue was added to each well in an amount equal to 10% of theculture volume and incubated for 3 h. The supernatants wereextracted and absorbance was measured at wavelengths of
570 nm and 600 nm.

Determination of Mineralization Levels
To determine the degree of calciﬁcation in MC3T3-E1
cells, cultured cells were stained with Alizarin red S.
Calciﬁcation levels were determined using a calcium assay
kit (Calcium C, Wako, Osaka, Japan). To quantitate theability of bone-nodule formation, mineralized bone nodules
were visible as white nodules after ﬁxing and were further
conﬁrmed by modiﬁed von Kossa staining (26). The contentsof each well were ﬁxed for 15 min in 10% neutral
buﬀered formalin, then a 5% silver nitrate solution (45 min)and 3% sodium thiosulphate (5 min) were used for von Kossa
staining.
Real time-PCR
Cultured MC3T3-E1 cells, with or without exposure to
H2O2, were homogenized using TRIzol reagent (InvtirogenCo., Carlsbad, CA), after which total RNA was isolated usinga FastPrepFP 120 Instrument (BIO 101 Inc., Vista, CA).
Samples (1 mg) of total RNA were then reverse transcribed,
followed by olig-dT primer and MuLV reverse transcriptase(ﬁnal volume; 20 ml), and then 5-ml aliquots from the diluted
cDNA (1/100-fold) were used as templates for real-time PCR.
To assess the eﬀects of H2O2 on the transcription of genes(listed in Table 1), PCR ampliﬁcation was performed using
SYBR Green real-time PCR master mix (TOYOBO Co.,
LTD, Osaka, Japan) with an ABI Prism 7700 and Sequence
Detection System software (Applied Biosystems, Foster City,
CA). The expression level of each gene was observed as thecycle number by real-time PCR, with their levels normalized tothat of the housekeeping enzyme glyceraldehygde-3-phosphate
dehydrogenase (GAPDH).
RESULTS
Effects of H2O2 on Mineralization of MC3T3-E1 Cells
Before attempting to clarify the eﬀects of H2O2 on theprocess of osteoblast diﬀerentiation, we tried to conﬁrm the
H2O2 eﬀects on cell viability of MC3T3-E1 cells. In order toinvestigate the eﬀect of osteoblastic diﬀerentiation in cellularsenescence caused by oxidative stress, we modiﬁed theexperimental condition by Chen et al. (25) and referred tothe study of Fatokun et al. (27). We assessed the viability of
MC3T3-E1 cells using Alamar Blue assay. As shown in
Table 2, the concentration of H2O2 more than 600 mM result

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ROS EFFECTS ON ANTIOXIDANT SYSTEM IN OSTEOBLAST DIFFERENTIATION

Table 1
Primers used for real time-PCR
Protein
name
NQO1

GenBankID

Primers

NM_008706 up;
down;
Nrf2
U70475
up;
down;
MafF
NM_010755 up
down;
MafG
NM_010756 up;
down;
MafK
NM_010757 up;
down;
Keap1
NM_016679 up;
down;
Runx2
NM_009820 up;
down;
OC
MMBGPR up;
down;
BSP
NM_008318 up;
down;
ALP
NM_007431 up;
down;
GAPDH M33197
up;
down;

50 -ctccaagcagcctgtttgacc-30
50 -tcccatcctcgtggagcaaag-30
50 -ttagtcagcgacagaaggac-30
50 -ccagttgaaactgagcgaaa-30
50 -gtggatcccttatccagcaaag-30
50 -catcagcgcttcatccga-30
50 -taaagtccaagacggatgct-30
50 -agaggagaaaggaagggaaa-30
50 -agctcatcatcgctaagaacagg-30
50 -gacagggcccgggttatg-30
50 -accagggcaagatctacgtc-30
50 -ggcccatggatttgagttctg-30
50 -ctcagtgatttagggcgcatt-30
50 -aggggtaagactggtcatagg-30
50 -agctatcagaccagtatggct-30
50 -ttttggagctgctgtgacatc-30
50 -taccgagcttatgaggacgaa-30
50 -gcatttgcggaaatcactctg-30
50 -ccatggtagattacgctcaca-30
50 -Atggaggattccagatacagg-30
50 -atcaccatcttccaggag-30
50 -atggactgtggtcatgag-30

The primers sets were designed using the primer searching software GenetyxMac Version 13.0.5. The suitableness of each primer set was conﬁrmed using
cDNA samples from ﬁrst strand cDNA, as described in ‘Materials andMethods’. The optimum temperature for ampliﬁcation was used to perform
real-time PCR, after conﬁrming the melting curve of each primer set.

Table 2Viability of MC3T3-E1 cells treated with H2O2
H2O2 (mM)
0
200
300
400
500
600
700

A570-A600

Cell viability (%)

0.390 + 0.031
0.396 + 0.031
0.390 + 0.031
0.390 + 0.004
0.376 + 0.036*0.265 + 0.049**0.190 + 0.033

100
101.5
100.0
100.0
96.4
67.9
48.7

MC3T3-E1 cells were treated with various concentrations of H2O2 for 3 h
and cell viability was assessed using AlamarBlue assay. Data are shown as
the mean + SD from three diﬀerent cultures. Signiﬁcant diﬀerencescompared to control are shown at the following probability level.
*P 5 0.05, **P 5 0.01.

in decrease of the viability signiﬁcantly, but the lower
concentration of H2O2 less than 500 mM did not have asigniﬁcant eﬀect on MC3T3-E1 cells on each culture period in29

our experimental condition. Therefore, we used a single exposure of 400 mM of H2O2 in the following experiments.
We attempted to examine the H2O2 eﬀects on the mineralization of long cultured MC3T3-E1 cells. After a single
treatment with H2O2, MC3T3-E1 cells were cultured for
50 days with a-MEM supplemented with 10% FBS, 5 mM of
b-GP, and 50 mg/ml of ascorbic acid. To determine the level ofmineralization, an Alizarin red S assay, a calcium assay, andvon Kossa staining were used. At 14-day, it has observed that
H2O2 induced the inhibitory eﬀect on calciﬁcation in
MC3T3-E1 cells (Fig. 1A). Next, we determined the alterationof calcium levels in long cultured MC3T3-E1 during 50 days
using a calcium assay kit. The cultured cells were washed with
Phosphate buﬀered saline (PBS) and calcium in the matrix was
dissolved with 0.5 M HCl. As shown in Figure 1B, the calcium
levels in H2O2-treated MC3T3-E1 cells reached a maximum at
30-day, while those in non-treated MC3T3-E1 cells remained
increased throughout the 50 days experimental period.
Calciﬁcation levels in the H2O2-treated MC3T3-E1 cells werehalf of those in the non-treated cells at all stages. In addition,
nodule formation in H2O2-treated MC3T3-E1 cells after
7, 14, 21, 30, 40, and 50 days was also reduced as comparedwith the non-treated cells, as shown by von Kossa staining(Fig. 1C).

Effects of H2O2 on Gene Expression of Antioxidant
Enzymes
The antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) is known to detoxify quinones and generate
antioxidant forms of ubiquinone. NQO1 is highly inducible bya number of stimuli, including electrophilic metabolites andoxidative stress (20, 28), and is considered to be a stressresponse indicator. To conﬁrm the results of antioxidant
system activity in MC3T3-E1 cells after treatment with H2O2,
we performed real time PCR analysis for the gene expressionof NQO1. Total RNA was extracted from both H2O2-treatedand non-treated MC3T3-E1 cells on days 1, 3, and 7. Eachreal-time PCR result was quantiﬁed and normalized to theexpression level of GAPDH. As shown in Figure 2, NQO1gene expression was strongly induced by H2O2 at ﬁrst day. The
increased gene expression of NQO1 remained high even at day
7, in spite of a single exposure of H2O2.
Nrf2 is responsible for the induction of NQO1 in responseto oxidative stress (21 – 23). Next, we determined the gene
expression for Nrf2, Keap1, Maf-F, Maf-G, and Maf-K using
real time-PCR analysis. As shown in Table 3, Nrf2 gene
expression was increased, whereas the up-regulation of Keap1
was not observed using the same cDNA samples (1/100-fold).
Effects of H2O2 on Gene Expression for Osteogenic
Marker Enzymes
The eﬀects of H2O2 on the gene expression of ALP, bonesialoprotein (BSP), osteocalcin (OC), and Runx2 were
determined using real-time and the end-point PCR assays.

ARAI ET AL.

Downloaded By: [Nihon University] At: 01:34 16 April 200830

Figure 1. Eﬀects of H2O2 on calciﬁcation of MC3T3-E1 cells. MC3T3-E1 cells were treated with 400 mM of H2O2 for 3 h. The
medium was immediately changed to fresh a-MEM to remove H2O2, then the cells were cultured for the indicated numbers ofdays. (A) H2O2-treated and non-treated MC3T3-E1 cells were cultured for 14 days and stained with Alizarin red S. (B) To
quantitate calcium levels, the cells were washed twice with PBS. Calcium in the matrix was dissolved with 0.5 M HCl, assay
reagent was added, and then the sample was measured at a wavelength of 598 nm. White and gray columns mean the results inthe non-treated and H2O2-treated MC3T3-E1 cells, respectively. *P 5 0.05, **P 5 0.01; signiﬁcant diﬀerence as compared to
the non-treated. Data are shown as the means +SE (n ¼ 5). (C) Eﬀects of H2O2 on nodule formation of MC3T3-E1 cells. Von
Kossa staining was used for determining mineralization.

The results of end-point PCR for ALP, BSP, Runx2, OC, Nrf2
and GAPDH after PCR of 38, 35, 45, 32, 35 and 35 cycles,
respectively, are shown in Figure 3. In H2O2-treated MC3T3E1 cells, ALP, BSP, Runx2 showed lower levels of expression
than in non-treated cells. Notable, the gene expression for
Runx2 was very low and strongly inhibited by H2O2
treatment. In contrast, that of OC was up-regulated by

H2O2 exposure from day 1. Nrf2 mRNA level was also
increased by H2O2.

DISCUSSION
Oxidative stress regulates cellular functions in multiple
pathological conditions, including bone formation byDownloaded By: [Nihon University] At: 01:34 16 April 2008

ROS EFFECTS ON ANTIOXIDANT SYSTEM IN OSTEOBLAST DIFFERENTIATION

Figure 2. Increased and maintained gene expression of NQO1.
Gene expression levels in H2O2-treated MC3T3-E1 cells werecompared with those of non-treated MC3T3-E1 cells. MC3T3E1 cells were treated with 400 mM of H2O2 for 3 h, then themedium was immediately changed to fresh a-MEM with
5 mM of b-GP and 50 mg/ml of ascorbic acid. The expression
level of each gene was observed as the cycle number by realtime PCR, with their levels normalized to that of thehousekeeping enzyme GAPDH. The solid column indicatesthe fold increases of gene expression levels for H2O2-treated
MC3T3-E1 cells compare with non-treated cells (opencolumn).

Table 3
Eﬀects of H2O2 on antioxidant related-gene expression on
day 7
Genes

Control

H2O2

Fold increase

GAPDH
NQO1
Nrf2
MafF
MafG
MafK
Keap1

1.0
235.9 + 77.4
231.2 + 66.2
0.75 + 0.34
n.d.
n.d.
n.d.

1.0
572.9 + 93.7
510.4 + 120.0
2.58 + 1.02
n.d.
n.d.
n.d.

2.43
2.21
3.44
–
–
–

Five microliters of 1/100-fold diluted cDNA was used as a template forreal-time PCR; n.d. ¼ ‘not detectable’ in this condition for 40 cycles.

osteoblastic cells. To clarify the eﬀects of H2O2 on the processof osteoblast diﬀerentiation leading to bone mineralization, asigniﬁcant end point in this process to create a mineralized
matrix in MC3T3-E1 cells was determined. We treated the31

Figure 3. End-point RT-PCR analysis of osteogenic marker
molecules. Ethidium bromide staining patterns for theampliﬁed PCR products using agarose gel electrophoresis
are shown. cDNA samples (1/100-fold) were used as
templates. Ampliﬁcation was done with 38, 35, 45, 32, 35,
and 35 cycles for ALP, BSP, Runx2, OC, Nrf2, and GAPDH,
respectively.

cells directly with H2O2 and found that their diﬀerentiation
was inhibited (Fig. 1). To clarify the eﬀects of oxidative stresson mineralization by MC3T3-E1 cells, we examined therelationship between the antioxidant system and bone formation in MC3T3-E1 cells. After a single exposure to H2O2, thelevels of calciﬁcation in H2O2-treated MC3T3-E1 cells werehalf that in non-treated cells at all culture stages. We
considered that if H2O2 could induce cell death signaling in
MC3T3-E1 cells, then the surviving cells in the plates would
proliferate ﬁrst, thus delaying the start of diﬀerentiation
toward the end-point of mineralization. As a result, calciﬁcation levels were expected to reach the same levels of the control
cells after a long culture period. However, even though
500 mM of H2O2 have no eﬀect on cell viability in our
experimental conditions (Table 2), the calciﬁcation level in
H2O2-treated MC3T3-E1 cells (400 mM of H2O2) was inhibited throughout the present 50 days experimental period(Fig. 1).
In previous studies, Nrf2 has been shown to be an essential
component of ARE-binding transcriptional machinery(29 – 31), and played an important role in regulating theexpression of many mammalian detoxifying and antioxidant
enzymes under oxidative or electrophilic stress in various
Nrf2-deﬁcient mice experiments. On the other hand, it wasalso reported that Nrf2 might negatively regulate osteoblastDownloaded By: [Nihon University] At: 01:34 16 April 2008

32

ARAI ET AL.

diﬀerentiation through inhibition of Runx2-dependent transcriptional activity in nrf2-overexpressed MC3T3-E1 cells (24).
In that study, the expression levels of Maf-G and Maf-F wereexpressed in a constitutive manner during cultivation from 3
to 28 days in nrf2-overexpressed MC3T3-E1 cells, withsustained expression seen with Runx2 mRNA for a periodof up to 28 days. In contrast, in our experiment, the gene
expression of Maf-F was up-regulated (Table 3) by a single
treatment with H2O2. Maf-F may partner with Nrf2 in
H2O2-stimulated MC3T3-E1 cells in antioxidant systems,
because gene expression for Maf-G and Maf-K was notdetectable after 40 cycles in our experimental condition. We
found that a single treatment with H2O2 induced thegene expression of Nrf2 and Maf-F in MC3T3-E1 cells(Table 3).
A previous study found that stable over expression of Nrf2
signiﬁcantly impaired Runx2-dependent stimulation of OC
promoter activity and recruitment of Runx2 on the OC
promoter without aﬀecting the expression of Runx2 mRNA
(24). Further, Nrf2 bound to the ARE-like sequence of the OC
promoter. As a result, they suggested that Nrf2 might
negatively regulate cellular diﬀerentiation through inhibitionof Runx2-dependent transcriptional activity in osteoblasts.
In the present H2O2-treated MC3T3-E1 cells, the gene
expression of Runx2 was inhibited (Fig. 3). We reasonably
expected that OC gene expression would be decreased by
H2O2-induced Runx2 reduction because of the mild ROS
stimuli. Unexpectedly, OC gene expression was increased by
the single exposure to H2O2 as compared with non-treated
MC3T3-E1 cells (Fig. 3). It is suggested that the increased gene
expression of Nrf2 and Maf-F, which are known to upregulate genes for antioxidant molecules, might have induced
the high expression level of the OC gene in the H2O2-treated
MC3T3-E1 cells.
A gene expression proﬁle during all phases of MC3T3-E1
cell diﬀerentiation (proliferation, matrix accumulation, andterminal diﬀerentiation indicated by mineralization) previously reported demonstrated the gene expression of OC
after 14 days (32). In the present H2O2-treated MC3T3-E1
cells, OC gene expression level was already high on day 1 andremained strongly induced on days 3 and 7, despite inhibitionof the gene expression of Runx2 (Fig. 3). We considered thatthe induction of OC gene expression during the early stages
might have caused an alteration of diﬀerentiation and/or
mineralization in MC3T3-E1 cells.
Our results indicated that a single exposure to
H2O2-induced inhibition of mineralization by the following
processes: (1) ROS inhibited the gene expression of osteogenic marker molecules in osteoblasts, particularly, Runx2,(2) the antioxidant system was maintained for a long period in
H2O2-treated MC3T3-E1 cells, and (3) Nrf2/MafF might alter
the gene expression proﬁle of some osteogenic marker
proteins. Therefore, this particular antioxidant system may
be a novel target with great beneﬁts for the discovery anddevelopment of innovative strategies useful for therapy andtreatment of a variety of diseases associated with cellular
damage caused by ROS, such as bone loss associated withaging.

ACKNOWLEDGEMENTS
This research was supported in part by a Grant from theAcademic Frontier Project for Private Universities, a matching fund subsidy from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), 2001 – 2005, andGrants-in-Aid for Scientiﬁc Research (A1; 16209063).
We gratefully acknowledge the collaboration with of
Dr T. Sato, Dr Y. Otsuka-Tanaka, and Dr J. Mega in thisstudy.

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BBRC
Biochemical and Biophysical Research Communications 349 (2006) 1050–1056
www.elsevier.com/locate/ybbrc

Eﬀect of F-spondin on cementoblastic diﬀerentiation of humanperiodontal ligament cells q
Masae Kitagawa a,b, Yasusei Kudo a, Shinji Iizuka a, Ikuko Ogawa b, Yoshimitsu Abiko c,
Mutsumi Miyauchi a, Takashi Takata a,*a

Department of Oral and Maxillofacial Pathobiology, Graduate School of Biomedical Sciences, Hiroshima University, Japanb
Center of Oral Clinical Examination, Hiroshima University Hospital, Hiroshima, 734-8553, Japanc
Department of Biochemistry, School of Dentistry at Matsudo, Nihon University, 270-8587, Japan
Received 21 August 2006
Available online 1 September 2006

Abstract
Cementum is a mineralized tissue produced by cementoblasts covering the roots of teeth that provides for the attachment of periodontal ligament to roots and surrounding alveolar bone. To study the mechanism of proliferation and diﬀerentiation of cementoblasts isimportant for understanding periodontal physiology and pathology including periodontal tissue regeneration. However, the detailed
mechanism of the proliferation and diﬀerentiation of human cementoblasts is still unclear. We previously established human cementoblast-like (HCEM) cell lines. We thought that comparing the transcriptional proﬁles of HCEM cells and human periodontal ligament(HPL) cells derived from the same teeth could be a good approach to identify genes that inﬂuence the nature of cementoblasts. We identiﬁed F-spondin as the gene demonstrating the high fold change expression in HCEM cells. Interestingly, F-spondin highly expressing
HPL cells showed similar phenotype of cementoblasts, such as up-regulation of mineralized-related genes. Overall, we identiﬁed F-spondin as a promoting factor for cementoblastic diﬀerentiation.
 2006 Elsevier Inc. All rights reserved.
Keywords: F-spondin; Cementoblast; Periodontal ligament; Diﬀerentiation

Cementum is a mineralized tissue produced by cementoblasts covering the roots of teeth that provides for theattachment of periodontal ligament to roots and surrounding alveolar bone [1]. Cementum contributes to the regeneration of the connective tissue attachment to root
surface, denuded due to periodontal disease. Therefore, it
is very important for studying the detailed mechanisms
proliferation and diﬀerentiation of human cementoblasts
to understand periodontal physiology and pathology,
including periodontal tissue regeneration. Several attempts
have been made to obtain makers of cementoblasts [2–5].
Recent studies have shown new makers of cementum orqThis work was supported in part by Grants-in-Aid from the ministry ofeducation, science and culture of Japan.
*
Corresponding author. Fax: +81 82 257 5619.
E-mail address: ttakata@hiroshima-u.ac.jp (T. Takata).
0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.08.142

cementum-periodontal ligament such as cementum-derived
attachment protein (CAP) [2] and cementum-derived protein (CP-23) [5]. However, the detailed role of these molecules has not been revealed in the diﬀerentiation of
cementoblasts.
We recently have established human cementoblast-like(HCEM) cell lines and human periodontal ligament(HPL) cell lines from same teeth by hTERT transfection
to examine the molecule involving with the diﬀerentiationof cementoblasts [6]. HCEM cell lines obtained from teeth
root lining cells showed high alkaline phosphatase (ALP)
activity, calciﬁed nodule formation, and the expression ofmineralized related genes, including type I collagen (COLI),
ALP, runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone sialoprotein (BSP), and CP-23. On theother hand, HPL cells from middle part of periodontal ligament showed low ALP and mineralization activity, andM. Kitagawa et al. / Biochemical and Biophysical Research Communications 349 (2006) 1050–1056

did not express OCN and BSP which are maker of genes
showing the mature diﬀerentiation of cementoblasts. Herewe compared the transcriptional proﬁles of HCEM cells
and HPL cells by microarray analysis in order to identify
the genes that diﬀer in their expression. We identiﬁed Fspondin as the gene demonstrating the high fold change
expression in HCEM cells. F-spondin is an extracellularmatrix protein required for pathﬁnding of commissuralaxons during ﬂoor plate development [7,8]. In the present
study, to know the role of F-spondin for cementoblastic differentiation, we transfected F-spondin into HPL cells, andexamined the expression of the mineralized related genes,
COLI, ALP, Runx2, OCN, BSP, and CP-23 in vitro.
Methods
These studies were performed in compliance with regulations administered by the experimentation committee of the Graduate School of
Biomedical Sciences, Hiroshima University.
Cell culture. HCEM cells which we previously established [6] andhuman osteoblasts (NHOst, OTT4, and Ost) provided by Dr. Tahara(Hiroshima University) were cultured in Minimum Essential Medium
Alpha (a-MEM, Invitrogen, Grand Island, NY) with 10% fetal bovine
serum (FBS) plus penicillin G solution (10 U/ml) and streptomycin(10 mg/ml) in a humidiﬁed atmosphere of 5% CO2 at 37 C. NHOst and
Ost are normal human osteoblast cells, OTT4 is immortalized humanosteoblast cell lines with SV40-T and hTERT. HPL cells were cultured in
Dulbecco’s Modiﬁed Eagle’s Medium (DMEM, Nissui Pharmaceutical
Co. Ltd.).
Gene array analysis. The human focus array using the system containing 8500 genes probes was used for comparing the transcriptional
proﬁles between HCEM cells and HPL cells. This array contains a broad
range of genes derived from publicly available, well-annotated mRNA
sequences. Total RNA was isolated from cultures of conﬂuent cells using
the RNeasy Mini Kit (Qiagen, K.K., Tokyo, Japan) according to the
manufacturer’s instructions. Preparations were quantiﬁed and their purity
was determined by standard spectrophometric methods.
Reverse transcription-polymerase chain reaction (RT-PCR). Total
RNA was isolated from cultures of conﬂuent cells using the RNeasy Mini
Kit (Qiagen) according to the manufacturer’s instructions. Preparations
were quantiﬁed and their purity was determined by standard spectrophotometric methods. cDNA was synthesized from 1 lg total RNA
according to the Rever Tra Dash (Toyobo Biochemicals, Tokyo, Japan).
The oligonucleotide RT-PCR primers for human F-spondin, rat F-spondin, chemokine orphan receptor (CMKOR1), phosphotriesterase related(PTER), solute carrier family membrane1(SLC14A1), and matrix
metalloproteinase (MMP) 13 are listed in Table 1. Primers for COLI,

1051

ALP, Runx2, OCN, BSP, CP-23, and glyceraldehyde-3-phosphate(GAPDH) were described previously [6]. Aliquots of total cDNA were
ampliﬁed with 1.25 U rTaq-DNA polymerase (Qiagen), and ampliﬁcations
were performed in a PC701 thermal cycler (Astec, Fukuoka, Japan) for
28–30 cycles after an initial 30 s denaturation at 94 C, annealed for 30 s at
55–60 C, and extended for 1 min at 72 C in all primers. The ampliﬁcation reaction products were resolved on 1.5% agarose/TAE gels (Nacalai
Tesque, Inc., Kyoto, Japan), electrophoresed at 100 mV, and visualized byethidium-bromide staining.
Generation of F-spondin highly expressing HPL cells. Packaging
GP-293 cells (Clontech, Palo Alto, CA) were transfected with retroviralplasmid encoding a rat F-spondin cDNA according to the manufacturer’s
instructions. A plasmid pMT21-FP5, encoding rat F-spondin cDNA, waskindly provided by Dr. Klar (Hebrew University). After 48 h of transfection, the virus-containing medium was collected and supplemented with
8 lg/ml polybrene (Sigma, St. Louis, MO). Then, the culture medium of
the target cells was replaced with this viral supernatant for 24 h. This
infection process was repeated a second time after a 12 h recovery innormal medium. The stable clones were obtained by puromycin selection(1 lg/ml) in the culture medium.
And clones were examined expressions of F-spondin by RT-PCR andWestern blot. Four F-spondin-expressing clones were chosen for thesubsequent experiments. A rat F-spondin cDNA was also cloned into
pBICEP-CMV-2 (Sigma) and was transfected into 293T cells.
Western blot analysis. Subconﬂuent cells, in 90 mm culture dishes, wereused for Western blot analysis. Western blotting was carried out as we
described previously [9]. Thirty lg/ml of protein was solubilized in Laemmli sample buﬀer by boiling, and subjected to 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed byelectroblotting onto a nitrocellulose ﬁlter. The ﬁlter was blocked for 1 h at
4 C with phosphate-buﬀered saline (PBS) buﬀer containing 5% nonfat
dried milk powder. Western blot analysis was performed using an antiSPON1 polyclonal antibody (ProSci, Flint Place Poway, CA), anti-FLAG
M2 monoclonal antibody (Sigma), and b-actin monoclonal antibody(Sigma) dissolved in PBS containing 5% nonfat dried milk powder andincubating for 60 min at room temperature. Incubation with a secondary
peroxidase-coupled goat anti-IgY Fc antibody (ProSci) and a secondary
peroxidase-coupled goat anti-mouse antibody was performed under thesame conditions. For detection of the immunocomplex, the ECL Western
blotting detection system (Amersham, Buckinghamshire, UK) was used.
Tissue samples. Tissue samples of periodontal tissues including toothwere retrieved from the Surgical Pathology Registry of Hiroshima
University Hospital, after approval by the Ethical Committee of our institutions. 3.7% buﬀered-formalin ﬁxed, decalciﬁed for 3 days, and paraﬃnembedded tissues were used for immunohistochemical examination.
Immunohistochemical staining. To examine expression of F-spondin inseven human periodontal tissues cases, the 4.5 lm sections were stained
immunohistochemically with an anti-SPON1 antibody (Prosci). Endogenous peroxidase was quenched by incubating with 0.3% H2O2 in methanol
for 30 min. Nonspeciﬁc staining was blocked using Dako Protein Block

Table 1
Oligonucleotide primer sequences utilized in the RT-PCR
RT-PCR primer set
F-spondin (human)
F-spondin (rat)
CMKOR1PTER
SLC14A1
MMP13

Sequence
F
R
F
R
F
R
F
R
F
R
F
R

0

GenBank Accession numbers
0

5 -ggaattggtccgagaagaca-3
5 0 -gggtttgttgggtgactcat-3 0
5 0 -gccgaagcttatgaggctatctcccgcg-3 0
5 0 -gccgctcgagctaacaagggtgcacgttgc-3 0
5 0 -acgtggtggtcttccttgtg-3 0
5 0 -agcatcaagacccgaagcta-3 0
5 0 -ttagccgagacacacagacg-3 0
5 0 -cgatcttcacagccctcttc-3 0
5 0 -cacgagtcatttctgcctga-3 0
5 0 -acacaagggatagggtgcag-3 0
5 0 -ctgagaggctccgagaaatg-3 0
5 0 -ggtccttggagtggtcaaga-3 0

NM_006108
NM_172067
NM_020311
NM_030664
NM_015865
NM_002427

1052

M. Kitagawa et al. / Biochemical and Biophysical Research Communications 349 (2006) 1050–1056

Serum Free (Dako, Carpinteria, CA). The sections were incubated withthe primary antibody (1:500) for overnight at 4 C, and then incubatedwith a secondary peroxidase-coupled goat anti-IgY Fc antibody (ProSci)
for 30 min. For visualization, they were treated with Liquid DAB (3,3 0 diaminobenzidine) Chromogen Syatem (Dako) according to the manufacturer’s protocol.
Measurement of ALP Activity. The quantitative analysis of ALP
activity was performed biochemically by Bessey-Lowry enzymologic
method using nitrophenyl phosphate as a substrate [10]. Cells were plated
in 24-well culture plates (1 · 105 cells per well) and cultured in DMEM
containing 10% FBS, penicillin G sodium (10 U/ml) and streptomycin
sulfate (10 mg/ml) for conﬂuent that cells were plated for 1week. The cells
were washed with PBS and homogenized ultrasonically in 0.5 ml of
10 mM Tris–HCl buﬀer (pH 7.4) containing 25 mM MgCl2. Aliquots of
the homogenates were used for quantiﬁcation of ALP activity.
Collagen assay. The measurement of collagen concentration was performed using Sircol Collagen Assay kit (Biocolor, Northern Ireland)
according to the manufacturer’s instruction. Cells were plated in 24-wellculture plates (1 · 105 cells per well), and cultured in DMEM containing
10% FBS, penicillin G sodium (10 U/ml) and streptomycin sulfate (10 mg/ml) for conﬂuent that cells were plated for 1 week. The culture media wereused for measurement of collagen concentration.
Statistical analysis. The results of cell growth analysis and quantitative
ALP activity were shown as means ± SE and analyzed for signiﬁcanceusing Wilcoxon’s test for non-paired examination. P values of less than
0.05 were judged to be statistically signiﬁcant.

Results
Identiﬁcation of F-spondin as a cementoblast-speciﬁc highly
expressed gene
We previously established a HCEM cell line from root
surface and a HPL cell line from middle part of periodontal
ligament of the same extracted human teeth [6]. In the present study, therefore, we thought that to compare the transcriptional proﬁles of HCEM cells and HPL cells could bea good approach to identify genes that inﬂuence the natureof cementoblasts (Fig. 1A). By microarray analysis, several
genes were selectively highly expressed in HCEM (Fig. 1A).
Among these genes, one of the highly expressed genes was
F-spondin. High expression of F-spondin in HCEM cells,
but not in HPL cells, was conﬁrmed by RT-PCR(Fig. 1B). As cementoblasts share many characteristics to

A

B

C

Fig. 1. F-spondin is identiﬁed as a cementoblast speciﬁc gene. (A) Human cementoblast-like (HCEM) cells and human periodontal ligament (HPL) cells
were established from the same extracted teeth by using modiﬁed enzymatic digestion methods. The transcriptional proﬁles of HCEM cells and HPL cells
were compared by microarray analysis. Highly expressed genes in HCEM cells are listed. Among these genes, F-spondin was identiﬁed the genedemonstrating the high fold change expression in HCEM cells. (B) The expression of F-spondin mRNA by RT-PCR. F-spondin mRNA expression isobserved in HCEM cells, but not in HPL cells. (C) The expression of F-spondin mRNA is not observed in human osteoblast cell lines (NHOst, OTT4 andOst). The expression of CMKOR1, PTER, and SLC14A1 is observed in both HCEM and NHOst.

M. Kitagawa et al. / Biochemical and Biophysical Research Communications 349 (2006) 1050–1056

osteoblasts, we examined the expression of F-spondin in
HCEM and human osteoblasts (NHOst, OTT4, and Ost).
Interestingly, HCEM expressed F-spondin mRNA, but
osteoblasts did not (Fig. 1C). On the other hand,
CMKOR1, PTER, and SLC14A1 were detected in both
HCEM and NHOst (Fig. 1C). Thus, F-spondin is speciﬁcally expressed in cementoblasts among the cells, which
compose the periodontal tissue.

A

B

F-spondin expression in human periodontal tissue
To conﬁrm the speciﬁc expression of F-spondin in
cementoblasts by immunohistochemical analysis, we ﬁrst
checked the accuracy of the antibody by Western blotting.
F-spondin antibody speciﬁcally recognized F-spondin
exprdession in FLAG-F-spondin transfected 293T cells(Fig. 2A). Then, we immunohistochemically examined its
expression in seve normal periodontal tissues. As expected,
F-spondin obviously expressed in the root lining cells(Fig. 2B).
High expression of F-spondin promotes the diﬀerentiation of
HPL cells in vitro
To know the role of F-spondin for cementoblastic diﬀerentiation, we stably transfected F-spondin into HPL cells,
which are the poorly diﬀerentiated cells in comparison withcementoblasts. We obtained 4 stable clones of F-spondin
highly expressing HPL cells (HPL-spondin) (Fig. 3A).
High expression of F-spondin changed the morphology,
showing short spindle shapes in comparison with control
cells (Fig. 3B). Next, we examined the expression ofmRNA for COLI, ALP, Runx2, OCN, BSP, and CP-23
by RT-PCR in F-spondin highly expressing HPL cells. In
addition, we examined collagen assay and ALP activity
by biochemical methods. Higher expression of ALP,

A

1053

Fig. 3. High expression of F-spondin in HPL cells. (A) Stable clones of
F-spondin highly expressing HPL-cells (HPL-spondin) expressed
F-spondin mRNA and protein. GAPDH and b-actin were used forloading control in RT-PCR and Western blot analysis. (B) HPL-spondin
cells show shorter spindle shapes (b) in comparison with control cells (a).
(scale bar = 100 lm).

OCN, and BSP, and lower expression of COLI wereobserved in F-spondin highly expressing HPL-cells in comparison with control cells (Fig. 4A). Both HPL-spondin
cells and control cells expressed Runx2 and CP-23 mRNAat the same levels (Fig. 4A). The ﬁndings that increased
expression of ALP mRNA and decreased expression of
COLI mRNA in F-spondin highly expressing HPL cells
were conﬁrmed by collagen assay (Fig. 4B) and ALP activity (Fig. 4C), respectively. To know the reason why type I
collagen decreased in HPL-spondin, we examined MMP13
expression. It is known that MMP13 is involved in type I
and II collagen degradation and expressed in both terminal

B

Fig. 2. F-spondin expression in human periodontal tissues. (A) Checking the accuracy of the anti-SPON1 antibody by Western blot. F-spondin antibody
speciﬁcally recognizes F-spondin in FLAG-F-spondin transfected 293T cells. (B) Immunohistochemical staining with the anti-SPON1 antibody in humanperiodontal tissue. High expression of F-spondin was observed in cell located along the root surface (triangles). C, cementum; D, dentin; PL, periodontal
ligament; AB, alveolar bone. Original magniﬁcation ·40 (a), ·200 (b).

1054

M. Kitagawa et al. / Biochemical and Biophysical Research Communications 349 (2006) 1050–1056

A

B

C

D

Fig. 4. F-spondin promotes the diﬀerentiation of HPL cells in vitro. (A) Expression of mRNA for COLI, ALP, Runx2, OCN, BSP, and CP-23 by
RT-PCR in HPL-spondin. Higher expression of ALP, OCN, and BSP, and lower expression of COLI are observed in HPL-spondin in comparison with
control cells. Both HPL-spondin and control cells express Runx2 and CP-23 mRNA at the same levels. (B) Mesurement of collagen concentration.
Collagen concentration tends to decrease in HPL-spondin by collagen assay. (C) ALP activity by biochemical methods. Increased ALP activity is observedin HPL-spondin cells in comparison with control cells. *Signiﬁcant diﬀerence (P < 0.05). (D) Expression of MMP13 mRNA by RT-PCR. HPL-spondin
cells express MMP13 mRNA at higher level in comparison with control cells.

hypertrophic chondorocytes and osteoblasts [11,12]. Interestingly, HPL-spondin cells expressed MMP13 mRNA at
higher level in comparison with control cells (Fig. 4D).
Discussion
The periodontium is a complex structural and functional
unit consisting of four diﬀerent components, i.e., gingiva,
alveolar bone, periodontal ligament, and cementum [13].
Cementum plays an important role of the attachment of
periodontal ligament to roots. The progenitor cells in bonemarrow spaces migrate into the perivascular area of theperiodontal ligament and move to the bone and tooth surface, and then diﬀerentiate into osteoblasts or cementoblasts [14,15]. In similar to osteoblasts, cementoblasts
express noncollagenous bone matrix proteins, COLI,
ALP, Runx2, OCN, BSP, and so on [15,16]. Recently,
CAP and CP-23 have been identiﬁed as new makers for
cementum or cementum-periodontal ligament [2,5]. However, we still do not know the detailed mechanism of
cementoblastic or osteoblastic diﬀerentiation from progen-itor cells in bone marrow spaces. To study the detailed
mechanism of cementoblastic diﬀerentiation and proliferation of cementoblasts, we recently established a humancementoblast-like cell line, HCEM, from root lining cells
and a human periodontal ligament cell line, HPL, frommiddle part of periodontal ligament of the same teeth byusing enzymatic digestion method [6].
In the present study, to identify the molecules playing
the role for cementoblastic diﬀerentiation, we comparedgene expression proﬁles between HCEM and HPL bymicroarray analysis. As HCEM and HPL were establishedfrom same teeth, we thought that comparing the gene
expression proﬁles between them could be a good approach
for identifying the gene of cementoblastic diﬀerentiation.
Here we found several genes highly expressed in HCEM
cells in comparison with HPL cells, but most genes except
for F-spondin expressed in osteoblasts by RT-PCR(Fig. 1A and C). Therefore, in the present study, we
focused on F-spondin. In fact, the expression of F-spondin
mRNA was observed only in cementoblast, but not in osteoblasts and periodontal ligament cells (Fig. 1B and C).

M. Kitagawa et al. / Biochemical and Biophysical Research Communications 349 (2006) 1050–1056

Moreover, F-spondin expression was observed only in thecells on the root surface (cementoblasts) by immunohistochemistry (Fig. 2B). These ﬁndings suggest that F-spondin
may be a speciﬁc molecule for cementoblasts among theperiodontal tissues.
F-spondin is an extracellular matrix protein and promotes neurite outgrowth of dorsal root ganglion cells[8,17] and spinal cord neurons in cell culture [18]. F-spondin promotes the diﬀerentiation of neural precursor cells tocells with the characteristics of neurons [19]. This is the ﬁrst
report on the function of F-spondin in cementoblasts. It isconsidered that periodontal ligament consists of diﬀerent
cell populations in various diﬀerentiation stages according
to the position in periodontal ligament. We previously
reported that cell populations with larger growth potential
were generally located in the middle position of periodontal
ligament and cell populations with higher ALP and mineralization activities toward the surface of the root in rat
periodontal ligament [20]. Therefore, we transfected
F-spondin into HPL cells which are more poor cell population than HCEM cells. Interestingly, high expression of
F-spondin changed the morphology (Fig. 3C) andincreased the expressions of ALP, OCN, and BSP mRNA,and ALP activity (Fig. 4A and C) in HPL cells, suggestingthat F-spondin may inﬂuence the diﬀerentiation of HPL
cells. As cementoblasts and osteoblasts expressed ALP,
OCN, and BSP, which play an important role for the mineralization [10], F-spondin promoted the diﬀerentiation of
HPL cells toward the mineralization. Moreover, both
HPL-spondin cells and control cells expressed CP-23
mRNA (Fig. 4A). CP-23 was expressed in mature cementum, cementoblasts, and periodontal ligament cells but
not osteoblasts [21]. Therefore, F-spondin promoted
cementoblastic diﬀerentiation not osteoblastic diﬀerentiation. We also found that high expression of F-spondin
decreased the expression of COLI mRNA and collagenconcentration (Fig. 4A and B). This ﬁnding may be caused
by increased expression of MMP13, because MMP13 is
known to be a collagenase of type I collagen and toincrease in the process of osteoblastic diﬀerentiation [22].
By microarray analysis, we could ﬁnd a lot of genes thathighly expressed in HCEM. For instance, CMKOR1,
PTER, and SLC14A1 are also highly expressed in HCEM.
Nuclear Factor I (NFIB), which is site speciﬁc DNA-binding protein [23], is also listed. In the future, we will examine
if these genes are involved in cementoblastic diﬀerentiation.
In summary, in the present study, we demonstrated a critical role of F-spondin for cementoblastic diﬀerentiation.
Our ﬁndings provide new and important information forunderstanding the mechanism of cementoblastic diﬀerentiation. We suggest that F-spondin could be used for a novel
molecular target for periodontal regeneration therapy.
Acknowledgments
We thank Dr. A. Klar for providing F-spondin vector.
We also thank Dr. H. Tahara for providing human osteo-

1055

blasts and Ms. A. Imaoka for supporting by microarray.
This work was supported by in part by Grants-in-Aid from
the Ministry of Education, Science and Culture of Japan to
M.K. (18791350), Y.A. (A1-16209063) and T.T.
(B1-1739048600).
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European Journal of Pharmacology 541 (2006) 198 – 204www.elsevier.com/locate/ejphar

An inhibitory effect of A20 on NF-κB activation in airway epithelium upon
influenza virus infection
Akira Onose a , Shu Hashimoto a,⁎, Shinichi Hayashi a , Shuichiro Maruoka a , Fumio Kumasawa a ,Kenji Mizumura a , Itsuro Jibiki a , Ken Matsumoto a , Yasuhiro Gon a , Tomoko Kobayashi a ,Noriaki Takahashi a , Yasuko Shibata b , Yoshimitsu Abiko b , Toshikatsu Shibata c ,Kazufumi Shimizu c , Takashi Horie aaDivision of Respiratory Medicine, Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-ku,
Tokyo 173-8610, Japanb
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo, Japanc
Department of Microbiology, Nihon University School of Medicine, Japan
Received 22 September 2005; received in revised form 15 March 2006; accepted 27 March 2006
Available online 20 May 2006

Abstract
Influenza is a major disease in humans. The reemergence of avian influenza A viruses has indicated that hyperinflammatory responses areclosely related to the severity of disease. Influenza virus infection induces nuclear transcription factor kappaB (NF-κB) activation. NF-κB and NFκB-dependent gene products promote lung inflammation and injury. Therefore, it is important to investigate the means to attenuate NF-κB
activation. A20 is a cytoplasmic zinc finger protein that inhibits NF-κB activity, However, little is known about the role of A20 in influenza virus
infection. Here, we have examined the role of A20 in influenza virus infection-induced NF-κB promoter activation in human bronchial epithelial
cells. The results showed that (1) A20 protein and mRNA are inducible and expressed in the lung from mice and human bronchial epithelial cells
upon influenza virus infection; (2) NF-κB promoter activation was induced in bronchial epithelial cells upon influenza virus infection; and (3)overexpression by transient transfection of A20 attenuated NF-κB promoter activation in bronchial epithelial cells. These results indicate that A20
may function as a negative regulator of NF-κB-mediated lung inflammation and injury upon influenza virus infection, thereby protecting the hostagainst inflammatory response to influenza virus infection.
© 2006 Elsevier B.V. All rights reserved.
Keywords: A20; Influenza virus; NF-κB; Bronchial epithelium; Inflammation

1. Introduction
Influenza virus infection is an acute respiratory infection andone of the most pandemic infectious diseases (Thompson et al.,2003). Highly pathogenic avian influenza virus subtypes H5N1
and H7N7 caused acute respiratory illness including fetal
disease (Shortridge, 2003). New strains of influenza A viruses
emerge in humans. Vaccine and anti-neuraminidase inhibitors
are widely used for the prevention and treatment of influenza⁎ Corresponding author. Tel.: +81 3 3972 8111; fax: +81 3 3972 2893.
E-mail address: shuh@med.nihon-u.ac.jp (S. Hashimoto).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.03.073

virus infection, but it is not logically possible to prepare
vaccines against all strains of influenza virus, especially
unexpected outbreak of non-human subtypes of influenza A
viruses (Webby and Webster, 2003; Cheng et al., 2004;
Gubareva et al., 2000). The most severe complications areviral pneumonia and lung injury (Yuen et al., 1998; Peiris et al.,
2004; Tran et al., 2004; Fouchier et al., 2004). During thedevelopment of viral pneumonia and lung injury, influenza
virus infection-associated proinflammatory responses are involved in the pathogenesis of viral pneumonia and lung injury(Peiris et al., 2004; Guan et al., 2004; Cheung et al., 2002; Van
Reeth, 2000). Therefore, it is important to clarify how toattenuate and terminate proinflammatory responses.

A. Onose et al. / European Journal of Pharmacology 541 (2006) 198–204

Activation of the nuclear transcription factor kappa-B (NFκB) is a hallmark of inflammatory processes induced byvarious stimuli, including viral pathogens (Fan et al., 2001;
Santoro et al., 2003). NF-κB activation during viral infection
has been interpreted as a protective response of the hostagainst viral infection (Santoro et al., 2003). NF-κB regulatesanti-viral cytokine, interferon (IFN) and tumor necrosis factor(TNF)-α against influenza virus infection (Santoro et al.,
2003; Seo and Webster, 2002; Julkunen et al., 2000). In
contrast, recent data have indicated that NF-κB activity is
associated with influenza virus infectivity and propagation(Nimmerjahn et al., 2004; Wurzer et al., 2004). Therefore, it
is important to investigate the regulatory mechanism in NFκB activity induced upon influenza virus infection. Preliminary data on comprehensive analysis of gene expression inhuman bronchial epithelial cells upon influenza virus
infection have shown that A20 mRNA expression is upregulated in bronchial epithelial cells upon influenza virus
infection.
The cytoplasmic zinc finger protein A20 is encoded by an
immediate early response gene and originally identified as a
TNF-α and interleukin (IL)-1-inducible gene product inendothelial cells (Song et al., 1996). A20 acts as an inhibitorof NF-κB activation and NF-κB-dependent gene expression(Gon et al., 2004; Hu et al., 1998). The expression of A20 isitself under the control of NF-κB (Krikos et al., 1992),
suggesting that A20 is involved in a negative feedback
regulation of NF-κB activation and NF-κB-dependent gene
expression. Therefore, it is possible that A20 may be a negative
regulator of influenza virus infection-induced inflammatory
responses. However, the role of A20 in inflammatory responses
has not been clarified. In order to do so, we examined the role of
A20 in NF-κB activation in human airway epithelial cells upon
influenza virus infectionin this study.
2. Materials and methods
2.1. Virus, mice and cells
Influenza virus A/Udon/307/72 (H3N2) and A/PR/8/34(H1N1) were grown as previously described (Kujime et al.,2000). Five- to six-week-old C57/BL mice were purchasedfrom Oriental Co., Inc. (Tokyo, Japan). Mice were housed inpathogen free conditions in accordance with the animal careguidelines of Nihon University School of Medicine. Bronchial
epithelial cell lines, NCI-H292, and human embryonic kidneycell lines, HEK293, were obtained from American Type Culture
as previously described (Gon et al., 2004).

199

were performed according to the manufacturer's instructions.
Prior to the hybridization to the HU-U95A Gene Chip, cRNAs
were first hybridized to the Test 3 chip to confirm thatdifferences in housekeeping genes between the samples wereless than 10%.
2.3. A20 mRNA and protein expression
For analysis of A20 mRNA expression in bronchial epithelial
cells, serum-starved bronchial epithelial cells were infected with
influenza virus (A/Udon/307/72) at moi of 5 and cultured. A20mRNA and G6PDH mRNA expression were determined at thetime indicated after influenza virus infection. For analysis of
A20 mRNA and protein expression in the lung from mice, themice were intranasally inoculated with 50 μl of 104 PFU of
influenza virus (A/PR/8/34) or 50 μl of PBS (control mice). The
mice were anesthetized and sacrificed, and the lungs were
removed at the desired times as indicated after infection. For
quantitative PCR analysis of mRNA expression, total mRNA
was isolated from BEC and homogenized lung from mice using
an RNA gents total RNA isolation system (Promega, Madison,
WI) and reverse transcribed with Improm-II reverse transcriptase (Promega). Primers for each A20 were designed using
Primer Express 2.0 software (Applied Biosystems, Foster City,
CA). The following oligonucleotides were used: human A20
sense, 5′-CTGCCCAGGAATGCTACAGATAC-3′; human A20
anti-sense, 5′-GTGGAACAGCTCGGATTTCAG-3′; mouse A20
sense, 5′-AAGCTTCTAAAGGAGTACTTGATAGTGATG-3′;
mouse A20 anti-sense, 5′-CAATTTTGCAGCGTTGATCAG-3′.
Quantitative RT-PCR was carried out by an ABI PRISM 7300
sequence detection system using SYBR Green PCR Master Mix(Applied Biosystems). Immunohistochemical analysis of A20
protein expression was performed using immunoperoxidase
technique as previously described (Koura et al., 2000). The
primary antibody was rabbit anti-human A20 polyclonal
antibody (Santa Cruz, Santa Cruz, CA) and nonimmune normal
rabbit IgG was used as a negative control. Counterstaining was
performed with haematoxylin.
2.4. Western blot analysis of IκB phoshorylation and total IκB
Serum-starved bronchial epithelial cells were infected with
influenza virus (A/Udon/307/72) at moi of 5 and were thencultured for desired times as indicated after infection with
influenza virus. Western blotting analysis of IκBα phosphorylation and degradation was performed using rabbit polyclonal
antibody to phosphorylated IκB (Promega) and rabbit polyclonal antibody to IκB (Promega). β̃ Tubulin was used as ahousekeeping protein.

2.2. Affymetrix gene chip analysis
2.5. Plasmids, transfection and luciferase reporter assay
Serum-starved bronchial epithelial cells were infected with
influenza virus (A/Udon/307/72) at multiplicity of infection(moi) of 5 and cultured for 6 h. Total RNA was isolated fromthe harvested cells using RNeasy Mini Kit (Qiagen, Valencia,
CA). Preparations of biotin-labeled cRNAs and hybridization
to the HU-U95A Gene Chip set (Affymetrix, Santa Clara, CA)

For analysis of NF-κB luciferase activity, serum-starved
bronchial epithelial cells, which had been transiently transfectedby NF-κB-Luc reporter plasmid, were infected with influenza
virus (A/Udon/307/72) at moi of 5 and were then cultured forthe desired time as indicated. For analysis of the effect of A20,

200

A. Onose et al. / European Journal of Pharmacology 541 (2006) 198–204

serum-starved bronchial epithelial cells were transiently
transfected with the pcDNA3-A20 expression vectors (Miller
et al., 1997) using FuGENE6 (Roche Diagnostics Corp.,
Indianapolis, IN). The total amount of cDNA was kept constantby supplementation with empty vector, pcDNA3 (Invitrogen,
Carlsbad, CA). Every transfection included 500 ng of NF-κBLuc reporter plasmid (Stratagene, La Jolla, CA) together with
5 ng of pRL-SV40-Renilla for normalization of transfection
efficiency. After 24 h with the transfection, bronchial epithelial
cells were infected with influenza virus (A/Udon/307/72) at moi
of 5 and NF-κB reporter activity was determined at the desired
times as indicated after infection with influenza virus aspreviously described (Jibiki et al., 2003).

Table 1

2.6. Virus protein synthesis

M55153
M24594

Influenza virus protein synthesis was determined by Western
blotting using the polyclonal anti-influenza virus serum aspreviously described (Maruoka et al., 2003). Briefly, HEK293
cells were transiently transfected with pcDNA3-Myc-A20(2 μg) or pcDNA3 (2 μg) as control. At 24 h after transfection,
the cells were infected with influenza virus (A/Udon/307/72) at
moi of 5 for the desired times as indicated. At the end ofcultivation, cells were then lysed and immunoblotted with
antibodies to polyclonal anti-influenza virus (1:1000 dilution
for overnight at 4) and anti-myc antibody (9E10) as previously
described (Maruoka et al., 2003). Anti-rabbit–horseradish
peroxidase antibody was used as a second antibody (1:2000
dilution for 1 h at room temperature). The antibody-antigen
complexes were detected using the ECL system (AmershamPharmacia Biotech). The appropriate expressions of Myc-A20
were confirmed in the lysate.

GeneBank
Gene description
accession no.

Uninfected IV infected Foldintensity
intensity
increase

AF008445
M30818446
735

10,992
16,876

24.6
23.0

61

1392

22.8

874
873
892
986

19,923
19,595
18,356
18,048

22.8
22.4
20.6
18.3

909

14,383

15.8

759
1090

10,685
15,211

14.1
14.0

629

7332

11.7

1063

11,878

11.2

195

2166

11.1

157

1738

11.1

424
1475
1065

4641
15,632
11,256

10.9
10.6
10.6

353

3717

10.5

951

9826

10.3

81

829

10.2

X04602
M33882
U52513
X02875
U72882M13755

U59877
M87434

D90070S82240
U18671
U77643
U15932
M59465

U51010
X138392.7. Statistical analysis
Statistical significance was analyzed by using analysis ofvariance (ANOVA). P value of less than 0.05 was consideredsignificant.
3. Results
3.1. Comprehensive gene expression in bronchial epithelial
cells upon influenza virus infection
For each gene on the chip-reading software makes anexpression call, such as “present”, “absent” or “marginal”
according to the difference between fluorescence intensity inperfectly matched sequences and mismatched sequences
containing a single base pair mutation. To minimize the numberof false positive results, we excluded the sequences whose
expression call of sample infected with influenza virus wasdetermined as “absent”. We were able to detect 5998 genes
among the 12,000 genes represented in the chip. We made threecriteria for the list that show genes induced by influenza virus
infection: (1) the intensity of expression was with at least a 10fold increase by influenza virus infection, (2) mean expression
intensity, determined by the average difference between

Phospholipid scramblase
Interferon-induced
cellular resistancemediator protein (MxB)
Interleukin BSF-2 (B-celldifferentiation factor)
p78 protein mRNA (MxA)
RIG-G mRNA
Oligo A synthetase E
Interferon-induced leucine
zipper protein (IFP35)
Interferon-induced
17-kDa/15-kDa protein
Transglutaminase (TGase)
Interferon-inducible
56-kDa protein
Low-Mr GTP-binding
protein (RAB31) mRNA
71-kDa 2 5 oligoadenylate
synthetase (p69 2-5A
synthetase)
ATL-derived
PMA-responsive(APR) peptide
RhoE = 26-kDa GTPasehomolog
Stat2 geneK12 protein precursor
Dual-specificity protein
phosphatase mRNA
Tumor necrosis
factor-α inducible
protein A20 mRNA
Nicotinamine
N-methyltansferase
Vascular smooth muscle
α-actinfluorescence in perfectly matched sequences and mismatchedsequences, in the sample infected with influenza virus, wasgreater than 100 (because low intense results were sometimes
inaccurate), and (3) expression difference between untreatedcells and influenza virus infected cells was statisticallysignificant. Table 1 represents the mRNA expression levelindicated as the average difference in only medium treated or
influenza virus infected cells. When using these criteria,
influenza virus infection was found to up-regulate theexpressions of 32 genes. Among these genes, A20 is aremarkable gene that inhibits NF-κB activity. Therefore, thestudy was conducted to examine the effect of A20 in NF-κB
activity induced upon influenza virus infection.
3.2. A20 mRNA expression in human bronchial epithelial cells
and lung
To verify the data on A20 mRNA expression obtained byGene Chip analysis, we quantitatively examined A20 mRNA
expression in bronchial epithelial cells and the lung from miceupon influenza virus infection using quantitative RT-PCR.

A. Onose et al. / European Journal of Pharmacology 541 (2006) 198–204

201

3.4. Influenza virus infection induces IκB phosphorylation and
NF-κB activation in human bronchial epithelial cells

Fig. 1. A20 mRNA expression in human bronchial epithelial cells and in lunghomogenates from mice. A20 mRNA expression in bronchial epithelial cells (A)and in the lungs from mice (B) were analyzed at times as indicated after
influenza virus infection by quantitave RT-PCR amplification. The results are
expressed as means ± S.D. of three different experiments. * indicates P < 0.05
compared with mRNA levels in influenza virus-uninfected bronchial epithelial
cells (A). * indicates P < 0.05 compared with mRNA levels in the lung frominfluenza virus-uninfected mice (B).

A20 mRNA expression in bronchial epithelial cells increased
at 8 h, sustained from 8 to 12 h, and thereafter decreased at
24 h (Fig. 1A). A20 mRNA expression in the homogenizedlung from mice increased at 12 h and was maximal at 24 h andthereafter decreased at 36 h (Fig. 1B). These results indicatethat A20 mRNA is inducible and expressed in the lung frommice and bronchial epithelial cells in a time-dependent
manner.

In the next series of experiments, we examined the effect of
A20 on NF-κB activity. To this end, we first examined NF-κB
signaling in bronchial epithelial cells upon influenza virus
infection. Among the many proteins exhibiting IκB function,IκBα is the only inhibitor that, in response to cell stimulation,
dissociates from the NF-κB heterodimer complex, withkinetics matching that of the translocation of NF-κB to the
nucleus. It has therefore been suggested that the inducible
activation of NF-κB is regulated mainly by the dissociation of
NF-κB an IκBα (Traenckner et al., 1995). In order to examine
influenza virus infection-induced NF-κB signaling, we
examined IκB phosphorylation and IκB degradation, and
NF-κB promoter activation in bronchial epithelial cells upon
influenza virus infection. As shown in Fig. 3A, amounts ofphosphorylated IκB in influenza virus-infected bronchial
epithelial cells increased at 4 h after influenza virus infection,
were maximal at 8 h and thereafter slightly decreased at 10 h(Fig. 3A, upper panel, P-IκBα). Amounts of total IκB protein
decreased along with time of culture periods, indicating an
increase in amounts of phosphorylated IκB proteins inversely
correlated with a decrease in amounts of total IκB proteins(Fig. 3A, middle panel, IκBα). Almost equal amounts of
tubulin protein were blotted with anti-tubulin antibody
regardless of the time of culture periods (Fig. 3A, lower
panel, β-tubulin). Next, the cells that had been transiently
transfected by NF-κB-Luc reporter plasmid were infected with
influenza virus and then NF-κB-dependent luciferase geneactivity was determined at various times as indicated after
infection with influenza virus. NF-κB reporter activity ininfluenza virus-infected cells increased at 4 h, was maximal
at 8 h and thereafter returned to the basal levels at 24 h(Fig. 3B).

3.3. A20 protein expression in the lungTo verify the data on A20 mRNA expression at the levelsof protein expression and identify the cellular source of A20,
we performed immunohistochemistry on lung tissue frommice upon influenza virus A/PR/8 infection. We stained lungtissue specimens with anti-A20 antibody. Representative
results are shown in Fig. 2. A20 is positively stained in the
bronchial epithelial cells and alveolar epithelial cells of lungspecimens from influenza virus-infected mice by anti-A20
antibody (Fig. 2A), but not in those from influenza virusuninfected mice (Fig. 2C). The lung tissue specimens of lungfrom either influenza virus infected or uninfected mice were
not stained with control antibody, normal rabbit IgG (Fig. 2B
and D).

Fig. 2. A20 expression in the lung tissue specimens from mice. Mice wereintranasallly inoculated with influenza virus or PBS (control mice) and the lungs
were removed on day 4. The lung specimens from influenza virus-infected mice
(A and B) and control mice (C and D) were stained with anti-A20 antibody (Aand C) or control rabbit IgG (B and D). Original magnification: ×400. The
results are representative of each group.

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A. Onose et al. / European Journal of Pharmacology 541 (2006) 198–204

Fig. 5. Influenza virus protein synthesis in A20-transfected HEK293. Influenza
virus protein synthesis was analyzed by Western blotting using polyclonal antiinfluenza virus antibody as described in Materials and methods. The results are
representative of two independent experiments.

Fig. 3. Influenza virus infection induces IκB phosphorylation and NF-κB
activation in human bronchial epithelial cells. IκBα phosphorylation (P-IκBα),
IκBα degradation (IκBα) and β-tubulin protein expression were analyzed attimes as indicated after influenza virus infection (A). NF-κB-dependent
luciferase gene activity was determined at times as indicated after influenza
virus infection (B). The results are expressed as means ± S.D. of three different
experiments. * indicates P < 0.05 compared with NF-κB activity in influenza
virus-uninfected cells.

3.5. Transfection of A20 attenuates influenza virus infectioninduced NF-κB promoter activation
Finally, we examined the effect of A20 in influenza virus
infection-induced NF-κB promoter activation. The bronchial
epithelial cells were transiently transfected by the A20
expression vector. Transient transfection of A20 attenuated

NF-κB promoter activation in bronchial epithelial cells infectedwith influenza virus (Fig. 4).
3.6. Influenza virus protein synthesis in A20-transfected cells
Transient transfection of A20 into bronchial epithelial cells
resulted in the inhibition of NF-κB promoter activation upon
influenza virus infection. If influenza virus growth is inhibitedin A20-transfected bronchial epithelial cells compared to that incontrol vector-transfected bronchial epithelial cells, the inhibited NF-κB activation might result from the inhibited replicationof influenza virus. To test this possibility, influenza virus protein
expression in A20-transfected HEK293 versus control vectortransfected HEK293 was compared. The amounts of tree major
influenza virus protein expression, including HA, NP and M1 in
A20-transfected HEK293, were comparable to those in control
vector-transfected HEK293, indicating that influenza virus
replication is not inhibited in A20-transfected (Fig. 5). These
results indicated that A20 doses not inhibit influenza virus
replication in these experimental conditions. The total numberof cells and cell viability determined by trypan blue exclusion
dye at the end of the culture period of each experiment(indicated in Figs. 1–5) did not differ with culture conditions(data not shown).
4. Discussion

Fig. 4. NF-κB activity is depressed in the A20-transfected human bronchial
epithelial cells. Bronchial epithelial cells were transiently co-transfected with thehuman NF-κB-Luc reporter plasmid as well as either the pcDNA-A20
expression vector or the empty pcDNA3 vector. After 24 h with the transfection,
bronchial epithelial cells were infected with influenza virus and NF-κB reporter
activity was determined at the desired times as indicated after influenza virus
infection. The results are expressed as means ± S.D. of three different
experiments. * indicates P < 0.05 compared with NF-κB promoter activity inempty pcDNA3 vector-transfected cells.

In this study, in order to clarify the potentially protective roleof A20 in lung inflammation and injury upon influenza virus
infection, we examined the inducible expression of A20 protein
and mRNA in the lung from mice and in bronchial epithelial
cells and the role of A20 in NF-κB promoter activation in
bronchial epithelial cells. The results showed that A20
expression was induced in the lung from mice and bronchial
epithelial cells, and overexpression of A20 attenuated NF-κB
promoter activation in bronchial epithelial cells upon influenza
virus infection. These results indicate that A20 may function asa negative regulator for inflammatory response in influenza
virus infection.

A. Onose et al. / European Journal of Pharmacology 541 (2006) 198–204

There are two major means to control influenza virus
infection: the inhibition of virus propagation and virus
infection-associated harmful events, including pulmonary
inflammation and injury. A20 is a cytoplasmic zinc finger
protein originally identified as a TNFα- and IL-1-inducible
gene product in endothelial cells (Song et al., 1996).
Subsequently, A20 expression is induced in a variety of cell
types upon various stimuli (Krikos et al., 1992; Becker et al.,
2000; Opipari et al., 1990; Jaattela et al., 1996). A20 negatively
regulates NF-κB signaling (Gon et al., 2004). However, the roleof A20 in influenza virus infection-induced NF-κB activity inbronchial epithelial cells has not been determined. Here, wehave shown that A20 is inducible and expressed in the lungfrom mice and in bronchial epithelial cells upon influenza virus
infection. To clarify the role of A20 in NF-κB activity, wetransiently transfected A20 into bronchial epithelial cells andexamined the role of overexpressed A20 in NF-κB promoter
activation in bronchial epithelial cells upon influenza virus
infection. The results have shown that overexpression of A20
results in the attenuation of influenza virus infection-induced
NF-κB promoter activation.
We must carefully interpret our results when extrapolating invitro data of the inhibitory effect of A20 on NF-κB activation to
the in vivo. Airway epithelial cells produce IFN-β and TNF-α
that exert anti-viral activity at the early stage of influenza virus
infection (Santoro et al., 2003; Seo and Webster, 2002; Julkunen
et al., 2000). They serve as the key player in the host defense
against influenza virus infection. The promoter of IFN-β geneand TNF-α gene contains an NF-κB-binding site and NF-κB
regulates IFN-β and TNF-α expression (Santoro et al., 2003;
Seo and Webster, 2002; Julkunen et al., 2000). Accumulated
cells including macrophages, T and B lymphocytes, naturalkiller cells and neutrophils at the site of viral infection arecapable of producing IFN-αγ, TNF-α, IL-1 and IL-18 (Kujime
et al., 2000; Tamura and Kurata, 2004). These cytokines
promote the development of Th1-type responses that are a
prerequisite for antiviral immunity. NF-κB regulates the gene
expression of these cytokines. Thus, NF-κB plays a defensiverole against influenza virus infection.
In the contrast, NF-κB is suggested to be harmful to the hostdefense against influenza virus infection (Nimmerjahn et al.,
2004; Wurzer et al., 2004; Ichiyama et al., 2004). NF-κB activity
enhances virus propagation (Wurzer et al., 2004). Alveolar type
II epithelial cells with low NF-κB activity were resistant to
influenza A virus infection, whereas the cells with high NF-κB
activity became susceptible to infection (Nimmerjahn et al.,2004). These studies indicate that the infectivity of influenza
virus is dependent on active NF-κB pathways. Influenza virusassociated encephalopathy is suggested to be a proinflammatory
disease and affected patients exhibit high morbidity andmortality. The levels of proinflammatory cytokines, such as
IL-6 in serum and NF-κB activity in peripheral bloodmononuclear cells, are closely associated with disease severity(Ichiyama et al., 2004). Avian influenza A virus infection causes
unusual severity of disease in humans. It has been indicated that
hyperproduction of NF-κB-dependent cytokines including
TNF-α and IP-10 may contribute to the unusual severity of203

human influenza H5N1 disease (Yuen et al., 1998; Peiris et al.,
2004; Tran et al., 2004; Fouchier et al., 2004). In the murine
model of viral pneumonia and lung injury upon influenza virus
infection, MIP-2 that is a murine counterpart of human IL8 increased in the lung tissue on day 2 after infection with
influenza virus accompanied with increased number of neutrophils in the lung (Ochiai et al., 1993). Neutralization of MIP-2
activity with anti-MIP-2 antibody or immunoglobulin preventsand attenuates lung injury and myocarditis upon influenza virus
infection (Kishimoto et al., 2004). Therefore, an inhibition of IL8 production is an important strategy to attenuate and minimize
pulmonary inflammation upon influenza virus infection. Taken
together, NF-κB activity might be harmful and should be downregulated when it is inappropriately and excessively activated.
Thus, NF-κB plays the host defense and also promotes
proinflammatory responses against influenza virus infection.
Vaccine is the key to controlling a pandemic and antiviraldrugs are effective in treating human influenza virus disease(Palese, 2004). Recently, short interfering RNAs (siRNA)
specific for highly conserved region influenza genes have beenshown to inhibit influenza virus A replication in the lung frommice infected with influenza A virus (Tompkins et al., 2004). In
addition to developing new antiviral drugs and vaccines, themeans to control hyperinflammatory responses in the host has
been expected. The inhibition of NF-κB by A20 may or may not
be advantageous to the host defense mechanism against
influenza virus infection. Recent data have indicated that A20
is critical to limiting inflammatory responses and host damages
in multiple tissues (Lee et al., 2000; Heyninck and Beyaert,
2005). In this study, we showed the negative regulatory role of
A20 in NF-κB activity in airway epithelial cell upon influenza
virus infection. A20 may be advantageous to the host defense
against influenza virus infection. However, the precise role of
A20 remains to be clarified.
Acknowledgement
This study was financially supported in part by a grant-in-aid
for High-Tech Research Center from the Japanese Ministry of
Education, Science, Sports and Culture to Nihon University,
General Scientific Research from the Ministry of Education ofJapan, and grants-in-aid for General Scientific Research from
the Ministry of Education of Japan (14570568, 15390260), andby Culture to Nihon University and Interdisciplinary General
Joint Research Grant for Nihon University.
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Lasers in Medical Science (2005) 20: 109–113
DOI 10.1007/s10103-005-0350-1

O R I GI N A L A R T IC L E

Yasuko Shibata Æ Naomi Ogura Æ Keisuke Yamashiro
Shogo Takashiba Æ Toshirou Kondoh Æ Keiji Miyazawa
Masaru Matsui Æ Yoshimitsu Abiko

Anti-inflammatory effect of linear polarized infrared irradiation
on interleukin-1b-induced chemokine production in MH7A
rheumatoid synovial cells
Received: 27 April 2005 / Revised: 10 May 2005 / Accepted: 6 June 2005 / Published online: 27 July 2005
 Springer-Verlag London Limited 2005

Abstract We examined the anti-inﬂammatory eﬀect of
infrared linear polarized light irradiation on the MH7A
rheumatoid ﬁbroblast-like synoviocytes (FLS) stimulatedwith the proinﬂammatory cytokine interleukin (IL)-1b.
Expression of messenger ribonucleic acids (mRNAs)
encoding IL-8, RANTES (regulated upon activation,
normal T cell expressed and secreted), growth-related
gene alpha (GROa), and macrophage inﬂammatory
protein-1a (MIP1a) was measured using real-time reverse
transcription polymerase chain reaction, and the secreted
proteins were measured in the conditioned media using
enzyme-linked immunosorbent assays. We found that
irradiation with linear polarized infrared light suppressed
IL-1b-induced expression of IL-8 mRNA and, correspondingly, the synthesis and release of IL-8 protein in

MH7A cells. This anti-inﬂammatory eﬀect was equivalent to that obtained with the glucocorticoid dexamethasone. Likewise, irradiation suppressed the IL-1binduced expression of RANTES and GROa mRNA.
These results suggest that the irradiation of the areas
around the articular surfaces of joints aﬀected by rheumatoid arthritis (RA) using linear polarized light may
represent a useful new approach to treatment.
Keywords Linear polarized light Æ Super Lizer ÆRheumatoid arthritis synoviocytes MH7A Æ IL-8 Æ
IL-1b Æ Dexamethasone

Introduction
Y. Shibata & Æ Y. Abiko
Department of Biochemistry and Molecular Biology,
Nihon University School of Dentistry at Matsudo,
2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan
E-mail: yshibata@mascat.nihon-u.ac.jp
Tel.: +81-47-3609328
Fax: +81-47-3609329
E-mail: yabiko@mascat.nihon-u.ac.jp
Tel.: +81-47-3609322
Fax: +81-473-3609329
N. Ogura Æ T. Kondoh
Department of Maxillofacial Surgery, Nihon University
School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi,
Matsudo, Chiba 271-8587, JapanK. Yamashiro Æ S. Takashiba
Department of Pathophysiology, Periodontal Science,
Okayama University Graduate Schoolof Medicine and Dentistry, 2-5-1, Shikata,
Okayama 700-8525, Japan
K. Miyazawa
Central Research Laboratories, Kissei Pharmaceutical Co., Ltd,4365-1, Hotaka, Minami-azumi, Nagano 399-8304, Japan
M. MatsuiTokyo Iken Co., Ltd., 1131-1, Higashi-Naganuma,
Inagi, Tokyo 206-0802, Japan

Rheumatoid arthritis (RA) is an autoimmune joint disease characterized by the inﬂammation and destructionof articular surfaces and bone. The pathogenesis of RA
involves the proliferation of synovial cells and theinvasion of joint cartilage and bone by ﬁbroblast-likesynoviocytes (FLS) and other inﬂammatory cells [1–3].
Indeed, the elastic synovial membrane is an initial targetof rheumatic inﬂammation and, along with chondrocytes, becomes a primary source of inﬂammatory factors(e.g., cytokines) that are secreted into the synovial ﬂuid
[4, 5]. For instance, levels of pro-inﬂammatory cytokines, such as interleukin (IL)-1b, are known to be elevated in the synovial ﬂuid of RA patients and to play a
key role in amplifying and perpetuating inﬂammationand joint destruction [6, 7].
The MH7A-immortalized rheumatoid FLS line wasestablished by stably transfecting FLS with the SV40 T
antigen gene. These cells grow more rapidly than theparental cells and, at this point, have attained over 150
population doublings [8]. The MH7A cells positively
stain for IL-1R, intercellular adhesion molecule-1(ICAM-1), CD16, CD40, CD80, and CD95 [8], and110IL-1b enhances the production of IL-6 and matrix metalloproteinases (MMPs), just as in the parental cells [9,10]. Thus, MH7A cells appear to be a useful model forinvestigating the regulation of rheumatoid FLS, the effects of IL-1b, and the potential eﬃcacy of new therapies
for treating RA.
Photodynamic laser therapy can be considered as a
new way to use arthroscopic techniques to treat
inﬂammation of the synovial membrane in RA [11].
Indeed, following gallium–aluminum–arsenide (Ga–Al–As) laser treatment, histological examination of theirradiated synovial membranes showed a ﬂattening ofepithelial cells, decreased villous proliferation, narrower vascular lumens, and lesser inﬁltration of
inﬂammatory cells than was seen in non-irradiatedsynovia [7]. An alternative approach makes use of thelinear polarized light instrument, which has been usedclinically with good eﬀect in several patients withinﬂammatory disease. It can be applied during physical therapy to relieve pain, and several studies haveshown that such irradiation relieves multiple-type
alopecia areata [12], improves the structure of stored
human erythrocytes [13], improves the ﬂexibility ofshoulder and ankle joints [14], and relieves temporomandibular joint pain [15, 16]. The biochemicalmechanism of action of linear polarized infrared irradiation is not yet clear, however.
Many cell types are now known to synthesize andrelease chemokines in response to injury, infection,
inﬂammation, and various other pathological conditions[17–20]. The aim of the present study was to determine
whether the proinﬂammatory cytokine IL-1b would
enhance chemokine production in MH7A cells serving
as a model of RA synovial cells, and, if so, whether the
linear polarized light irradiation would suppress that

Fig. 1a, b An output spectrum from Super Lizer after polarizing
plate passage. An output spectrum from Super Lizer (a) was
measured by the spectrometer (Yokogawa Electric Co., Tokyo,
Japan). The light from a halogen lamp (150 W) is input to a lowpass ﬁlter having a ﬁlm designed to ﬁlter out waves of wavelength
shorter than 600 nm on a quartz glass. The transmitted light is
input to the bundle ﬁber, then next to square glass pillar, and then aconvex lens. Finally, the transmitted light is irradiated to dishes
through the polarizing plate in the CO2 incubator (b). The intensityof the light power was measured using a power meter (thermo-

eﬀect, thereby, establishing a potential role for linear
polarized infrared irradiation in the clinical treatment ofinﬂamed synovial tissue in RA joints.

Materials and methods
Human rheumatoid arthritis model
The MH7A cells, which were derived from human FLS
isolated from the knee joint of an RA patient and retain
the morphologic and functional characteristics of primary synovial cells [8], were obtained from Cell Bank,
Riken Bioresource Center (Ibaraki, Japan). The cells
were maintained in RPMI-1640 medium (Life Technologies Inc., Grand Island, NY, USA), supplemented with
10% fetal bovine serum and penicillin-streptomycin at
37C under a 95% air/5% CO2 atmosphere. For
experimentation, MH7A cells were ﬁrst incubated for
16 h in medium containing 10% fetal calf serum andthen in fresh medium containing recombinant humanIL-1b (CalBiochem, EMD Bioscience Inc., Darmstadt,
Germany).
Linear polarized infrared irradiation
A Super Lizer (HA2200, Tokyo Iken Co., Ltd., Tokyo, Japan) linear polarized light instrument was used
to irradiate cells at wavelengths ranging from 600 nm
to 1,600 nm, as shown in Fig. 1a. Dishes containing
IL-1b-stimulated MH7A cells were irradiated for 685 s
in the CO2 incubator (Fig. 1b). The depth and intensity of the irradiation were regulated by respectively
varying their distance from the laser source and thecouple type) (NEOARK Co., Tokyo, Japan). Irradiation occupancy was ‘‘0.5,’’ because the turn on/oﬀ of light was every second.
The energy density was calculated as follows: (energy density)=(average power of density)·(exposure time). The irradiation
area of the laser was 45 cm2 (6.7·6.7 cm). Energy density=0.0056 W (average power of density)·685 s (exposuretime)=3.8 J/cm2 [average power=0.5 W·0.5 (occupancy; lamp
lighting time/exposure time)=0.25 W, average power of den(irradiationsity=0.25 W(average
power)/45 cm2area)=0.0056 W]

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Immunoassays
Levels of IL-6, tumor necrosis factor a (TNF-a), IL-6,
IL-8, and RANTES (regulated upon activation, normal
T cell expressed and secreted) protein in media conditioned by MH7A cells grown on 35-mm2 plates wereassayed using commercially available ELISAs (EndogenInc., Woburn, MA, USA).

Real-time kinetic RT-PCR
To assess the eﬀects of irradiation on the transcriptionof genes encoding IL-8, RANTES, growth-related genealpha (GRO-a), macrophage inﬂammatory protein-1a(MIP1a), and the housekeeping enzyme glyceraldehyde3-phosphate dehydrogenase (GAPDH), MH7A cells
grown to 70% conﬂuence on 100-mm2 plates were
irradiated for 2 h using a Super Lizer or left untreated,
after which, they were homogenized using TRIzol Reagent (Life Technologies, Gaithersburg, MD, USA),and the total RNA was isolated using a FasRNA kit
with a FastPreoFP 120 Instrument (BIO 101 Inc., Vista,
CA, USA). The level of expression of speciﬁc mRNAs
was determined using an ABI Prism 7700 and sequence
detection system software (Applied Biosystems, Foster
City, CA, USA). The primer sets used for real-time kinetic RT-PCR (polymerase chain reaction) were as follows: for IL-8, 5¢-ATC ACT TCC AAG CTG GCC
GTG GCT-3¢ (forward) and 5¢-TCT CAG CCC TCTTCA AAA ACT TCT C-3¢ (reverse) [21]; for RANTES,
5¢-TAC ACC AGT GGC AAG TGC TC-3¢ (forward)
and 5¢-GAA GCC TCC CAA GCT AGG AC-3¢ (reverse); for GRO-a, 5¢-TGC AGG GAA TTC ACC CCA
AG-3¢ (forward) and 5¢-CAG GGC CTC CTT CAGGAA CA-3¢ (reverse); for MIP1a, 5¢-TTT GGT GTC
ATC ACC AGC AT-3¢ (forward) and 5¢-GCC TGAAAC AGC TTC CAC TC-3¢ (reverse); and for GAPDH, 5¢-ATC ACC ATC TTC CAG GAG-3¢ (forward)
and 5¢-ATG GAC TGT GGT CAT GAG-3¢ (reverse).

Results
Anti-inﬂammatory treatment reduces IL-8 production
by MH7A cells
The importance of interleukins such as IL-1, IL-6, IL-8,
and TNF-a in RA was initially proposed on the basis ofanalysis of gene regulation at the local site of the disease,

**

3000

Production of IL-6 and IL-8 (pg/dish)duration of the irradiation. For experimentation, weused pulsed irradiation at a frequency of 1 Hz, which
produced an energy density of 3.8 J/cm2. The cells
were harvested immediately after irradiation, and theribonucleic acid (RNA) was extracted. In addition, theconditioned media were collected and stored at 20C
for later use.

2500

2000

*
1500

1000

**

*

500

**
0
0

0.5

1

IL-1β concentration (unit/ml)
Fig. 2 Dose-dependent synthesis of IL-8 (open circles) and IL-6(ﬁlled circles) in MH7A cells treated with IL-1b. The cells wereincubated with the indicated concentrations of IL-1b for 3 h. The
results are expressed as mean±SD (n=4); * P<0.05, ** P<0.005
versus control medium without IL-1b

the synovium. IL-8 is the prototypical member of asuperfamily of small (8–10 kDa), inducible, secreted
chemoattractant cytokines (chemokines) that wereoriginally discovered as monocyte-derived factors
capable of attracting and activating neutrophils [17–19,
22]. Chemokines, including IL-8, have the function of
being key mediators in diverse inﬂammatory disorders
via promoting the recruitment, proliferation, and activation of vascular and immune cells. It has been foundFig. 3 Inhibitory eﬀect of linear polarized infrared irradiation usinga Super Lizer on the production of IL-8 in MH7A cells. After the
addition of a low dose of IL-1b (0.1 units/ml), the cells were
irradiated using a Super Lizer (3.8 J/cm2) or administered DEX(1 lM) and incubated for 3 h in a CO2 incubator: hatched bars with
IL-1b, open bars without IL-1b, None without anti-inﬂammatory
treatment, SL irradiation using a Super Lizer, DEX treatment with
1 lM dexamethasone. The results are expressed as mean±SD(n=5); * P<0.05, ** P<0.005 versus control medium without IL-1b

112
Table 1 Chemokine gene expression in MH7A cells stimulated with IL-1b
None

IL-8
RANTES
GRO
MIP1a

SL

DEX

None

IL-1b

None

IL-1b

None

IL-1b

100
100
100
100

925
173
555
179

91
103
60
68

535
118
340
155

92
122
43
289

109
116
179
287

Eﬀects of irradiation with linear polarized light using a Super Lizer and administration of dexamethasone on the expression of chemokine
mRNAs in MH7A cells plated on 100-mm2 dishes at 1.5·105 cells/dish. After incubating for 16 h, fresh medium containing 0.1 U/ml IL1b was provided, Super Lizer irradiation or DEX was applied, and the incubation was continued for an additional 3 h. The RNA
extraction was carried out in a clean box using a FasRNA kit. Five independent extractions were carried out, after which, the extracted
RNAs were pooled, measured, and reverse-transcribed. Using real-time kinetic RT-PCR, the expression of several chemokine mRNAs
were detected, and their levels were normalized to that of the housekeeping enzyme GAPDH. The data shown are mean percentages
normalized to control dishes, which were assigned a value of 100%: None no anti-inﬂammatory therapy, SL irradiation with a Super Lizer(3.8 J/cm2), DEX treatment with dexamethasone (ﬁnal concentration, 1 lM)

that IL-8 is the major chemoattractant in RA synovial
tissues [23]. By assaying conditioned media using speciﬁc
ELISAs, in the present study, we were able to conﬁrm
that IL-1b dose-dependently stimulates IL-8 production
and release by MH7A cells (Fig. 2). Production of another chemokine, RANTES, and that of cytokines,
TNF-a, and IL-6, were also measured in IL-1b-stimulated MH7A cells. A smaller amount of IL-6 than IL-8
was detected from MH7A cells into the medium. The
RANTES and TNF-a productions from MH7A cells
were not found by the ELISA method (data not shown).
We then evaluated the eﬀect of irradiating the cellswith linear polarized infrared light using a Super Lizeron the IL-1b-stimulated production and secretion of IL8. We found that, after the MH7A cells were stimulatedwith a low dose (0.1 units/ml) of IL-1b, irradiation reduced the production of IL-8 to 24.4% of that seen in
non-irradiated control cells. Notably, this was equivalent to the inhibitory eﬀect obtained with the antiinﬂammatory glucocorticoid dexamethasone (DEX)(Fig. 3).
Expression of chemokine mRNA in IL-1b-stimulated
MH7A cells
Finally, we examined the relative levels of chemokine
mRNA expression in MH7A cells using real-time kinetic
RT-PCR. We found that IL-1b induced about a ninefold increase in the expression of IL-8 mRNA, and that
infrared Super Lizer irradiation suppressed this eﬀect of
IL-1b, though not quite to the degree that DEX did(Table 1). In addition, although we detected no releaseof RANTES protein from cells by ELISA, the levels of
its mRNA were increased 1.7-fold by IL-1b stimulation,and Super Lizer irradiation and DEX suppressed thateﬀect to similar degrees. Expression of GROa, with andwithout IL-1b stimulation, was also suppressed by both
irradiation and dexamethasone, whereas the expressionof MIP1a mRNA appears to have been suppressedsomewhat by Super Lizer irradiation, but not by DEX.

Discussion
It was recently reported that irradiation with linear
polarized light at near-infrared wavelengths using theSuper Lizer is an eﬀective short-term treatment in patients with RA-aﬀected temporomandibular pain [15, 16].
However, very little is known about the molecularmechanism by which linear polarized light exerts thiseﬀect. In the present study, therefore, we examined theeﬀect of infrared Super Lizer irradiation on IL-1b-induced production of the chemokine IL-8 in the MH7A
cultured cell line. The IL-8 levels are known to be elevatedin such inﬂammatory diseases as RA, osteoarthritis,
osteomyelitis, and periodontal disease [24]. And althoughmacrophages, neutrophils, and endothelial cells are allconsidered to be primary sources of IL-8, we found thatIL-1b stimulated the immortalized FLS to express IL-8mRNA and to secrete high levels of IL-8 protein.
Many anti-rheumatic drugs provide relief from RA
by acting as immunomodulators [25–30], though someexert an inhibitory eﬀect on various cytokines [27, 28]. In
that regard, glucocorticoids (e.g., DEX) reportedly
downregulate the expression of several inﬂammatory
genes, including those encoding IL-1, TNF-a, IL-6, andIL-8 [31]. We investigated whether infrared Super Lizer
irradiation could inﬂuence gene transcription in MH7A
cells under basal conditions and following stimulation
with IL-1b, and found that the eﬀect of this irradiationon the synthesis and release of IL-8 protein paralleled its
eﬀect on the expression of IL-8 mRNA (Table 1).
Likewise, the irradiation aﬀected the expression of both
RANTES and GROa mRNA, though the relationshipbetween gene transcription and protein synthesis remains unclear in those cases. It thus appears that Super
Lizer irradiation exerts eﬀects on MH7A cells that are
similar to those of DEX and are consistent with therapeutic eﬃcacy in RA. Since RA is a chronic inﬂammatory disease, steroid administration was often in thelong-term treatment. Our results demonstrate that thelinear polarized infrared irradiation kept the MH7A

113

cells to their normal state after the stimulation withproimﬂammatory cytokine, IL-1b, due to the suppressed
mRNAs expression of chemokines, as DEX did.
Revealing the anti-inﬂammatory mechanism of the linear polarized infrared irradiation in rheumatoid FLS
will contribute to the future treatment of RA patients.
Acknowledgments This research was supported in part by a grantfrom the ‘‘Academic Frontier’’ Project for Private Universities:
matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) 2001–2005 and Grants-inAid for Scientiﬁc Research (A1; 16209063, A2; 13357017).

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