Mol Cell Biochem (2010) 335:263–272
DOI 10.1007/s11010-009-0276-1
Activation of RhoA and FAK induces ERK-mediated osteopontin
expression in mechanical force-subjected periodontal ligament
fibroblasts
So-Yeon Hong • Young-Mi Jeon • Hyun-Jung Lee
Jong-Ghee Kim • Jin-A. Baek • Jeong-Chae Lee
Received: 4 June 2009 / Accepted: 16 September 2009 / Published online: 2 October 2009
Ó Springer Science+Business Media, LLC. 2009
Abstract The precise mechanism by which Rho kinase
translates the mechanical signals into OPN up-regulation in
force-exposed fibroblasts has not been elucidated. Human
periodontal ligament fibroblasts (hPLFs) were exposed to
mechanical force by centrifuging the culture plates at a
magnitude of 50 g/cm2 for 60 min. At various times of the
force application, they were processed for analyzing cell
viability, trypan blue exclusion, and OPN expression at
protein and RNA levels. Cellular mechanism(s) of the
force-induced OPN up-regulation was also examined using
various kinase inhibitors or antisense oligonucleotides
specific to mechanosensitive factors. Centrifugal force up-
regulated OPN expression and induced a rapid and tran-
sient increase in the phosphorylation of focal adhesion
kinase (FAK), extracellular signal-regulated kinase (ERK),
and Elk1. Pharmacological blockade of RhoA/Rho-asso-
ciated coiled coil-containing kinase (ROCK) signaling
markedly reduced force-induced FAK and ERK1/2 phos-
phorylation. Transfecting hPLFs with FAK antisense oli-
gonucleotide diminished ERK1/2 activation and force-
induced OPN expression. Further, ERK inhibitor inhibited
S.-Y. Hong
Department of Orthodontics, Dental Clinic, School of Medicine,
Ajou University, Suwon 443-721, South Korea
Y.-M. Jeon
H.-J. Lee
J.-G. Kim
J.-A. Baek
J.-C. Lee (&)
Institute of Oral Biosciences and 21 Century Education Center
for Advanced Public Dental Health (BK 21 Program), School
of Dentistry, Chonbuk National University, Jeonju 561-756,
South Korea
e-mail: leejc88@chonbuk.ac.kr
J.-C. Lee
Department of Bioactive Materials and Research Center
of Bioactive Materials, Chonbuk National University,
Jeonju 561-756, South Korea
•
significantly OPN expression, Elk1 phosphorylation, and
activator protein-1 (AP-1)-DNA binding activation, but not
FAK phosphorylation, in the force-applied cells. These
results demonstrate that FAK signaling plays critical roles
in force-induced OPN expression in hPLFs through inter-
action with Rho/ROCK as upstream effectors and ERK-
Elk1/ERK-c-Fos as downstream effectors.
Keywords Centrifugal force

Human periodontal ligament fibroblasts
Osteopontin

Mechanotransduction
Focal adhesion kinase

Extracellular signal-regulated kinase
Introduction
Periodontal ligament (PDL) is composed of various types
of cells and extracellular matrix components. Human PDL
fibroblasts (hPLFs) are the most abundant cells present in
PDL and maintain PDL integrity [1, 2]. hPLFs also regu-
late alveolar bone formation and resorption stimulated by
physiological and mechanical stimuli [3], phemomena that
have been associated with the ability of hPLFs to produce
various bone-related cytokines and enzymes, such as
receptor activator of nuclear factor-kappaB (NF-jB) ligand
(RANKL) [4], osteoprotegerin (OPG) [5], interleukin (IL)-
1b, and tumor necrosis factor (TNF)-a [6]. Therefore,
alveolar bone remodeling during orthodontic tooth move-
ment is believed to be greatly affected by the expression
levels of these proteins produced by hPLFs in the PDL.
The major processes of bone remodeling are controlled by
the expression of various bone tissue components. Osteo-
pontin (OPN), one of the main bone-related components, is a
non-collagenous, glycosylated phosphoprotein [7] known to
be produced by both osteoblasts and osteoclasts [8, 9]. The
123
264
expression of OPN increases in response to mechanical
stress, a process that is required for mineralization in bone
cells and tissues [10–12]. OPN also plays important roles in
tissue inflammation by regulating the adhesion, attachment,
and spreading of osteoclasts to the bone surface [13–15].
Based on these observations, it is believed that OPN is
expressed by a mechanosensitive gene in bone cells and is a
critical factor in regulating bone formation and resorption in
responses to mechanical stimuli.
Specific expression of OPN in periodontal tissues has
been reported. OPN is expressed mainly in hPLFs and is
also present in resorption lacunae in the PDL [16, 17]. OPN
expression is regulated by growth factors and/or matrix
proteins [18, 19], and is closely related to the presence of
periodontal disease [20, 21]. Based on previous reports that
mechanical force causes a periodontal inflammatory
response with an attendant remodeling of alveolar bone, it
is clear that OPN plays an important role in the regulation
of alveolar bone remodeling after orthodontic treatment.
A recent report showed that OPN expression at both the
mRNA and protein levels increased significantly in hPLFs
in a mechanical force-dependent manner, and this OPN up-
regulation was markedly inhibited by a Rho kinase inhib-
itor [22]. OPN up-regulation via Rho kinase also appeared
to be correlated with the force-induced release of adenosine
triphosphate [23]. These findings suggest that mechanical
force increases OPN, with Rho kinase playing a critical
role; however, the precise role of Rho kinase in OPN up-
regulation in mechanically stressed hPLFs is unclear. The
downstream signaling mediators of Rho kinase in mechan-
ical force-exposed hPLFs are also unknown.
Focal adhesion kinase (FAK), a tyrosine kinase linked to
integrin signaling, is known to be a major mechanosensitive
kinase that is rapidly activated by mechanical stimuli in
cardiac myocytes [24, 25]. Considerable evidence supports a
critical role for FAK in the regulation of genes sensitive to
mechanical stress [26, 27]. More importantly, FAK activa-
tion is dependent on Rho kinase-mediated signaling in
mechanical force-exposed cells [28–30]. Our recent findings
also showed that centrifugal force up-regulates OPN
expression in human gingival fibroblasts through extracel-
lular signal-regulated kinase (ERK)-mediated signaling
[12]. These findings led us to hypothesize that Rho-FAK-
ERK-mediated signaling has an indispensable role in OPN
up-regulation in mechanical force exposed-hPLFs.
In this study, we therefore examined whether Rho kinase
signaling is required for FAK activation in centrifugal
force-exposed hPLFs. We also determined the exact roles
of Rho kinase/FAK signaling on OPN expression by
inhibiting Rho kinase and FAK signaling using specific
inhibitors or transfection with antisense oligonucleotides.
Furthermore, we investigated the roles of mitogen-
activated protein kinases (MAPK) on the expression of OPN.
123
Mol Cell Biochem (2010) 335:263–272
Materials and methods
Chemicals
Unless otherwise specified, all chemicals were obtained
from Sigma Chemical Co. (St. Louis, MO, USA). MAPK
inhibitors, PD98059 (2-(2-amino-3-methoxyphenyl)-4H-
1-benzopyran-4-one), SB203580 (4-(4-fluorophenyl)-2-
(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), and
SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), were pur-
chased from Tocris Biosciences (MO, USA). Protein
kinase C (PKC) inhibitor (GF1092023X; 3-[1-[3-(dimeth-
ylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyr-
role-2,5-dione) was purchased from Upstate (Chicago, IL,
USA). RhoA inhibitor (Clostridium botulinum C3 exoen-
zyme; C3-Exo) and Rho-associated coiled coil-containing
kinase (ROCK) inhibitor (Y-27632) were obtained from
Calbiochem (EMD Biosciences, San Diego, CA, USA).
The chemicals were dissolved in sterilized distilled water,
absolute ethanol or dimethylsulfoxide (DMSO) prior to
use. The final ethanol or DMSO concentration did not
exceed 0.5% (v/v) in any experiment.
Cell culture
hPLFs were obtained from three healthy male individuals,
20–30 years of age, who had undergone premolar extrac-
tion for orthodontic treatment. hPLFs were cultured
according to the methods described elsewhere with minor
modifications [31]. Written informed consent for use of the
periodontal tissues was obtained from all donors. This
study was approved by the Review Board of Chonbuk
National University Hospital (CNUH). Briefly, teeth were
washed with phosphate buffered saline, and the PDL were
carefully removed from the middle third of the root. After
enzymatic digestion and dissection, cell suspensions were
incubated in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS; Hyclone,
Logan, UT, USA) and antibiotics (100 IU/ml penicillin G
and 100 lg/ml streptomycin) in 100-mm culture dishes.
All the cultures were maintained at 37°C under a humidi-
fied gas mixture of 5% CO2/95% air and were exchanged
with fresh medium every 3 days. When the cells had
reached confluence, they were subcultured for several
passages and spread onto various types of flat-bottomed
culture plates before applying a centrifugal force. All the
experiments were carried out at passages 4 through 7.
Application of mechanical force
hPLF cultures in this study were exposed to a mechanical
force by the application of centrifugal force to the cells, a
well-established in vitro compression model for cultured
Mol Cell Biochem (2010) 335:263–272
cells [32] that is useful for evaluating the direct effects of
compression on cells without side effects [33]. hPLFs were
also treated with various kinase inhibitors for 1 h prior to
centrifugation of culture plates using a horizontal micro-
plate rotor (Universal 32 R, Hettich, Germany) at a mag-
nitude of 50 g/cm2 for 60 min as described previously [32].
The applied force was calculated based on the following
equation: P = (m 9 r 9 rpm2 9 p2)/(A 9 9.8 9 900),
where P is the kg pressure per cm2 of cells, m is the mass of
medium (g), r is the radius (0.12 m), rpm is revolution per
min, and A is the area of contact between the medium and
cells (cm2). hPLFs were subjected to analyses to help
determine the mechanisms involved in the force-induced
up-regulation of OPN at various times after treatment with
the applied force.
Measurement of SDH activity
3-(4,5-Dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium
bromide (MTT) was used to evaluate the succinate dehy-
drogenase (SDH) activity of HGF. In this assay, only living
cells take up the yellow MTT, which is then converted to a
dark blue product by an enzyme, SDH, present in mito-
chondria. The concentration of this blue product is pro-
portional to the SDH activity that reduces MTT, and is also
closely related to the viability of the cells. Twenty-four
hours after application of a centrifugal force of magnitude
(0–150 g/cm2) for 60 min, 5 ll of MTT solution (5 mg/ml
in PBS as stock solution) were added to each well, and the
cells were incubated for a further 4 h at 37°C. After
removing the culture media, acidic isopropanol (70 ll) was
added to each well, and the absorbance of the plates was
read at 570 nm using a SpectraCountTM ELISA reader
(Packard Instrument Co. Downers Grove, IL, USA).
Trypan blue exclusion assay
The trypan blue exclusion assay was used to determine the
level of cytotoxicity caused by applying a centrifugal force
to the cells. Twenty-four hours after application of a force
of magnitude 0–150 g/cm2 for 60 min, the cells were
stained with 0.4% trypan blue and approximately 100 cells
were counted for each treatment. The level of cytotoxicity
was calculated as follows: % cytotoxicity = [(total
cells - viable cells)/total cells] 9 100%.
Western blot analysis
Whole cell lysates from the control and force-exposed
hPLFs were prepared in NP-40 lysis buffer (30 mM Tris–
Cl, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 1% NP-40).
Protein content was quantified using the Bradford method
[34]. Equal amounts of protein extracts were separated by
265
12% SDS-PAGE and blotted onto polyvinyl difluoride
membranes. The blots were probed with primary antibodies
overnight at 4°C prior to incubation with secondary anti-
body in a blocking buffer for 1 h. The blots were developed
with enhanced chemiluminescence reagents (Amersham
Pharmacia Biotech Inc., Buckinghamshire, UK) and were
exposed to X-ray film (Eastman-Kodak, Rochester, NY,
USA). The polyclonal antibodies specific to extracellular
signal-regulated kinase (ERK; sc-94) p-FAK (sc-16662),
FAK (sc-558), OPN (sc-20788), and actin (sc-7210), and
the monoclonal antibodies specific to p-ERK (sc-7383) and
p-Elk-1 (sc-8406) were purchased from Santa Cruz Bio-
technology (Santa Cruz, CA, USA).
Reverse transcription-PCR (RT-PCR)
Total RNA was extracted according to the manufacturer’s
instructions (SV Total RNA Isolation System, Promega,
Madison, WI, USA). Reverse transcription and PCR
amplification were also performed using an Access RT-
PCR System (Promega) according to the manufacturer’s
protocol. Primer sequences used to amplify OPN are as
follows: OPN, 50 -GCC ATG ACC ACA TGG ATG AT-30
(forward) and 50 -ATG GCC TTG TAT GCA CCA TT-30
(reverse). The housekeeping gene, glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) with the primer
sequences 50 -AAC CTG CCA AAT ATG ATG AC-30
(forward) and 50 -ATA CCA GGA AAT GAG CTT GA-30
(reverse), was used as a positive control. PCR was per-
formed for 30–35 cycles at 94°C for 30 s, at 50–55°C for
30 s, and at 72°C for 1 min in a DNA Thermal cycler
(PTC-100, Waltham, MA, USA). PCR products were
analyzed on a 1.5% agarose gel and visualized by ethidium
bromide staining. Band intensity was calculated using a gel
imaging system (F1-F2 Fuses type T2A, Bio-Rad, Segrate,
Italy).
Transfection with FAK antisense oligonucleotides
To determine the role of FAK signaling in OPN expression
of compressive force-exposed hPLFs, the cells were seeded
at 1 9 106 cells/well in 6-well plates and transfected with
antisense oligonucleotides (ASO) specific to FAK or non-
sense oligonucleotides (NSO). The ASO targeting FAK
(sense sequences: ATG TGC TGG GCT AAA GAA;
antisense sequences: TTC TTT AGC CCA GCA CAT) and
NSO (50 -ATA ATC GAC GTT CAA GCA-30 ) as a control
were selected from the human FAK gene (GenBank
Accession No. L13616) and were synthesized by Cosmo
Co. Ltd (Seoul, South Korea). Each transfection with oli-
gonucleotides was performed using LipofectamineTM 2000
Transfection Reagent (Invitrogen, MD, USA) according to
the manufacturer’s protocol with slight modifications.
123
266
Briefly, the transfection was performed in DMEM/serum
and antibiotic-free medium with the lipofectamine reagent
at a final concentration of 1 lM at 37°C for 6 h. Culture
medium was then replaced with lipofectamine-free DMEM
containing 10% FBS. After 24 h, centrifugal force was
applied to the cells and they were then processed for the
analyses of FAK expression, ERK phosphorylation, and
OPN expression.
Electrophoretic mobility shift assay (EMSA)
hPLFs grown in 6-well plates were treated with an ERK or
RhoA specific inhibitor 1 h before the application of cen-
trifugal force, and 2 h thereafter, nuclear proteins from the
cells were prepared as described elsewhere [35]. Activator
protein (AP)-1 binding activity for a specific DNA
sequence was determined by EMSA. Briefly, DNA-protein
binding reactions were carried out for 30 min at room
temperature using 10–15 lg of the protein in a 20-ll vol-
ume containing 1 lg/ml albumin, 0.5 lg/ll poly (dI-dC),
5% glycerol, 1 mM DTT, 1 mM PMSF, 10 mM Tris–Cl
(pH 7.5), 50 mM NaCl, 5 mM MgCl2, 30,000 cpm of
[a-32P] dCTP-labeled oligonucleotides, and the Klenow
fragment of DNA polymerase. The samples were separated
on 6% polyacrylamide gels, dried, and exposed to X-ray
films. The following oligonucleotide primer sequences
were used for AP-1: 50 -AAG GGA TCC GGC TGA CTC
ATC ACT AG-30 and 30 -CTA GGC CGA CTG AGT AGT
GAT CGG AA-50 .
Statistical analyses
Unless otherwise specified, all data are expressed as the
means ± standard deviation (SD) from three independent
experiments. A one-way ANOVA was used for multiple
comparisons using SPSS version 15.0 software. A P value
\0.05 was considered statistically significant.
Results
Centrifugal force up-regulates OPN at the mRNA
and protein level in hPLFs
To examine how compressive force influences the
expression of OPN in hPLFs, the cells were exposed to
centrifugal force at various magnitudes from 0 to 150 g/cm2
for 60 min. At various times after the force, the cells were
subjected to RT-PCR (Fig. 1a), Western blot analysis
(Fig. 1b), trypan blue staining (Fig. 1c), and MTT assays
(Fig. 1d). In parallel with the previous study [12], this
study shows that both the mRNA and protein levels of OPN
vary according to the magnitude of the force applied. The
123
Mol Cell Biochem (2010) 335:263–272
Fig. 1 Effects of centrifugal force on OPN induction, cytotoxicity,
and SDH activity in hPLFs. hPLFs were exposed to centrifugal force
at various magnitudes from 0 to 150 g/cm2 for 60 min. a At 4 h after
the applied force, the cells were subjected to RT-PCR. In addition,
24 h after the applied force, the cells were subjected to Western blot
analysis (b), trypan blue staining (c), and MTT assays (d), respec-
tively. The data from Western blot analysis and RT-PCR were
obtained from at least three independent assays and were quantified
by densitometry after normalizing the bands to GAPDH or actin.
* P \ 0.01, ** P \ 0.01, and *** P \ 0.001 versus the control
values. TB trypan blue
expression of OPN increased at 20 g/cm2 and peaked at
50 g/cm2 of applied force, whereas a force more than
100 g/cm2 did not stimulate the expression of OPN. OPN
expression also increased depending on the length of time
for the applied force, such that the increase of OPN started
after 1 h and peaked at 4 h after application of force (data
not shown). Centrifugal forces of less than 100 g/cm2 for
60 min caused neither significant cytotoxicity nor reduced
SDH activity (Fig. 1c, d). In contrast, exposing hPLFs to a
force greater than 100 g/cm2 for the same length of time
induced significant cytotoxicity with a decrease in mito-
chondrial activity. Therefore, we used a magnitude of 50 g/cm2
and 60 min as the model force and time period, respec-
tively, in subsequent experiments. These model conditions
did not cause morphological changes in the hPLFs (data
not shown).
ERK-mediated signaling is critical to the force-induced
up-regulation of OPN
MAPKs play an important role in the signal transduction of
mechanotransduction in osteoblastic cells [36]. We previ-
ously reported that centrifugal force induces a rapid and
transient activation of MAPKs in human gingival fibro-
blasts [12] as well as in hPLFs [37]. Therefore, the first
goal of the present study was to determine the roles of
Mol Cell Biochem (2010) 335:263–272
Fig. 2 Effects of MAPK and PKC inhibitors on OPN expression in
centrifugal force-exposed hPLFs. hPLFs were treated with 10 lM of
MAPK or PKC inhibitor 1 h before exposure of the cells to force
(50 g/cm2/60 min). OPN mRNA levels were determined by RT-PCR
after 4 h of applied force. The results were obtained from three
independent assays and were quantified by densitometry after
normalizing the bands to GAPDH. ** P \ 0.01 and *** P \ 0.001
versus the control values. ## P \ 0.01 versus the experiments
Fig. 3 Effects of MAPK and PKC inhibitors on Elk1 phosphoryla-
tion in centrifugal force-exposed hPLFs. hPLFs were treated with
10 lM of MAPK or PKC inhibitor 1 h before exposure of the cells to
centrifugal force (50 g/cm2/60 min). After 1 h, protein samples were
prepared from the force-exposed cells, and the level of p-Elk1 was
measured by Western blot analysis. The result obtained from three
independent assays was quantified by densitometry after normalizing
the band to actin. ** P \ 0.01 and *** P \ 0.001 versus the control
values. ## P \ 0.01 versus the experiments
MAPKs in the cytoplasmic signaling pathways involved in
the force-induced up-regulation of OPN in hPLFs.
We initially treated the cells with 10 lM of MAPK or
PKC inhibitors 1 h before exposure to centrifugal force,
and then determined the OPN mRNA level through RT-
PCR. Figure 2 shows the relative OPN mRNA levels in
hPLFs cultured in the presence and absence of inhibitors.
No inhibitors used in this study affected basal OPN mRNA
levels. Treatment of hPLFs with SP600125, SB203580, or
GF109203X did not decrease OPN mRNA levels that had
been increased by the applied force. However, treating the
267
cells with PD98059 significantly inhibited OPN expression,
thereby suggesting that the activation of ERK is critical to
the force-induced expression of OPN.
The ERK inhibitor alone also significantly reduced the
cellular level of p-Elk1 that had been increased by applica-
tion of force (Fig. 3). Because Elk1 is one of the Ets family of
transcription factors activated by ERK phosphorylation, and
the OPN promoter contains three Ets-binding sites [38],
these results suggest that ERK-Elk1-mediated signals are
important downstream molecules responsible for the up-
regulation of OPN in force-exposed hPLFs.
FAK and ERK activation after centrifugal force
is dependent on RhoA/ROCK signaling
In subsequent experiments, we focused on examining the
hypothesis that RhoA-ROCK-FAK signaling cascades are
critical to OPN up-regulation in force-exposed hPLFs. This
hypothesis was based on the observations that a compressive
force induces the expression of OPN through Rho kinase-
mediated signaling [22], and that FAK is a critical me-
chanotransducer that is activated by the RhoA/ROCK
pathway in cardiac myocytes [24]. We first examined whe-
ther centrifugal force affects the levels of FAK and p-FAK
using Western blot analysis (Fig. 4). Centrifugal force
induced rapid and sustained FAK activation in the cells, such
that the increase of p-FAK started at 30 min and was main-
tained up to 2 h after application of force. The level of whole
FAK protein did not change during the experiment.
We next examined the precise roles of Rho and ROCK on
the phosphorylation of FAK and ERK that had been
increased by centrifugal force. To this end, hPLFs were
pretreated with 0.5 lM of the RhoA selective inhibitor, C3
exoenzyme, or 10 lM of the ROCK inhibitor, Y27632, for
1 h and were then subjected to centrifugal force. Pretreat-
ment of the cells with C3 exoenzyme had no effect on basal
levels of FAK and ERK phosphorylation (Fig. 5). In contrast,
the exoenzyme significantly inhibited the force-induced
increases of p-FAK and p-ERK. Similarly, treating the cells
with Y-27632 reduced the force-mediated phosphorylation
of FAK and ERK to the basal levels, while Y-27632 itself
had no effect on the basal levels of p-FAK and p-ERK as
compared to unforced control cells. These results support the
involvement of RhoA/ROCK signaling in mechanical force-
induced FAK and ERK activation in hPLFs.
FAK is a key downstream mediator of RhoA/ROCK
signaling involved in OPN expression in centrifugal
force-exposed hPLFs
We further examined the role of FAK on centrifugal force-
induced OPN expression by transfecting hPLFs with non-
sense (NSO) or antisense oligonucleotides (ASO). The data
123
268 Mol Cell Biochem (2010) 335:263–272

Fig. 4 Centrifugal force induces rapid and sustained FAK phosphor-

ylation in hPLFs. hPLFs were exposed to a centrifugal force of 50 g/cm2
for 60 min, and at the indicated times after application of the force,
protein samples were prepared from the cells to analyze FAK
phosphorylation. The data obtained from three independent assays were
quantified by densitometry after normalizing the bands to FAK.
** P \ 0.01 versus the control cells (no force applied)
Fig. 6 Inhibition of FAK by transfection with antisense oligonucle-
otides dramatically reduces the force-induced increases in the protein
levels of p-FAK and p-ERK, and the protein and mRNA levels of
OPN. hPLFs were transfected with FAK antisense oligonucleotides
and 24 h thereafter they were exposed to a centrifugal force of 50 g/cm2
for 60 min. At various times after the application of force, protein and
RNA samples were prepared to determine p-FAK, p-ERK, and OPN
protein levels through immunoblotting, and OPN mRNA levels
through RT-PCR. The data obtained from triplicate experiments were
quantified by densitometry after normalizing the bands to total FAK
and ERK1/2, GAPDH or actin. * P \ 0.05, ** P \ 0.01, and
*** P \ 0.001 versus the control values. # P \ 0.05, ## P \ 0.01,
and ### P \ 0.001 versus the force-exposed and NSO-transfected
cells. NSO nonsense oligonucleotides, ASO antisense oligonucleotides

from immunoblotting and RT-PCR analyses revealed that

transfection with FAK ASO but not NSO significantly
suppressed the force-mediated activation of FAK and ERK
(Fig. 6, left panels) as well as the up-regulation of OPN at
both the mRNA and protein levels (Fig. 6, right panel).
Transfection with either FAK ASO or NSO did not affect
the basal levels of FAK, ERK, and OPN at either the
mRNA expression or protein induction levels.

RhoA and ERK inhibitors inhibit force-induced

AP-1 DNA binding and OPN expression
Fig. 5 Treating hPLFs with RhoA or ROCK inhibitors significantly
diminishes the force-induced phosphorylation of both FAK and ERK.
MAPKs have roles as upstream effectors of AP-1 by
hPLFs were treated with 0.5 lM RhoA inhibitor C3 exoenzyme or
10 lM ROCK inhibitor Y27632 for 1 h prior to exposure to force mediating the activation of c-Jun and c-Fos proteins which
(50 g/cm2/60 min). At 1 h after the application of force, protein bind to the promoter region of mechanosensitive genes [39,
samples were prepared and p-FAK and p-ERK levels were deter- 40]. Centrifugal force-induced up-regulation of OPN in
mined by Western blot analysis. The data obtained from triplicate
human gingival fibroblasts has also been reported to be
experiments were quantified by densitometry after normalizing the
bands to total FAK and ERK1/2. ** P \ 0.01 versus control values. associated with the ERK-AP-1-mediated pathway [12].
#
P \ 0.05 and ## P \ 0.01 versus control values Figure 7a shows the binding activity of AP-1 to a specific

123
Mol Cell Biochem (2010) 335:263–272
Fig. 7 Treatment of hPLFs with ERK, RhoA or ROCK inhibitor
inhibits the force-induced increases of AP-1-DNA binding activity
and OPN expression, but not of p-FAK. hPLFs were treated with
10 lM PD98059, 0.5 lM C3 exoenzyme or 10 lM Y-27632 for 1 h
before exposure to centrifugal force (50 g/cm2/60 min). At 2 h after
the application of force, the cells were processed for EMSA (a) and
DNA sequence in the presence and absence of PD98059 or
C3 exoenzyme. The force-induced increase of AP-1 DNA
binding was markedly reduced after pretreatment with
either an ERK or RhoA inhibitor, thereby supporting the
participation of AP-1-mediated downstream signaling in
force-induced OPN up-regulation.
In order to more clarify the roles of RhoA/ROCK-
mediated signaling in OPN up-regulation, the cells exposed
to centrifugal force in the presence of C3-Exo or Y-27632
were processed for RT-PCR. The result showed that the
force-mediated increase of OPN expression was dramati-
cally suppressed by the inhibitors (Fig. 7b). Moreover,
ERK inhibitor did not affect FAK phosphorylation that was
stimulated by centrifugal force (Fig. 7c), indicating that
FAK acts as upstream effecter of ERK-mediated signaling.
Discussion
The most widely used initiators of mechanotransduction
signaling are integrin receptors [30]. Numerous studies
have highlighted the important roles of integrins in mech-
anotransduction in cells [30, 41, 42]. Integrin-mediated
signaling mainly includes the downstream activation of
adaptor proteins such as FAK, Rho family proteins, pax-
illin, and caveolin [25, 43]. Similar to FAK, Rho proteins
are quite sensitive to mechanical stimuli and play a critical
role in various cellular events in response to these stimuli
through interactions with integrins [44–46]. Therefore, we
suggest that among the various mechanoreceptors, inte-
grins act as the main upstream receptors of Rho and FAK,
although additional experiments showing a direct interac-
tion between integrins and these adaptor proteins are
necessary.
269
RT-PCR (b). c In addition, at 1 h after the force, the levels of p-FAK
and FAK in the cells were analyzed by Western blotting. The data
obtained from triplicate experiments were quantified by densitometry
after normalizing the bands to GAPDH or total FAK. * P \ 0.05,
** P \ 0.01, and *** P \ 0.001 versus the control values
Rho proteins also interact with other downstream
effectors to transmit mechanical signaling into the nucleus.
ROCK, a member of a family of serine/threonine kinases,
is the most well-characterized Rho effecter protein. ROCK
transmit various mechanical and physiological stimuli into
the nucleus through downstream effectors, and is also
involved in the regulation of various cellular events [29,
30, 47]. Thus, the Rho/ROCK-mediated signaling pathway
is considered critical for transducing a broad range of
mechanical signals within cells [24, 48]. Direct evidence
suggesting the involvement of Rho kinase in the mechan-
ical stress-induced expression of OPN in hPLFs was first
provided by the finding that OPN up-regulation by con-
tinuous compressive force in hPLFs was prominently
inhibited by a Rho kinase inhibitor but not by cytochalasin
B [22]. Our current findings also show the participation of
the RhoA/ROCK pathway, as demonstrated by the dra-
matic reduction of p-FAK and p-ERK in cells treated with
the selective inhibitors, C3 exoenzyme or Y-27632.
Oligonucleotide array analysis revealed that mechano-
transduction in hPLFs includes Rho/ROCK-dependent
pathways, although another pathway may also compensate
for the effect of the Rho/ROCK pathway on the expression
of mechanosensitive genes in these cells [49]. In addition,
an important role for Rho kinase in the regulation of OPN
expression has been reported in other cell types [50]. These
previous findings in combination with our results strongly
suggest that RhoA/ROCK signaling plays a crucial role in
transducing mechanical signals that are responsible for the
induction of OPN in force-exposed hPLFs.
Considerable data showing the involvement of FAK
activation as a downstream target of RhoA/ROCK signal-
ing in many cell types suggest that cascade pathways
via integrins-Rho/ROCK-FAK signaling are critical to
123
270
mechanotransduction in cells. However, the precise
mechanisms by which Rho/ROCK signaling activates
downstream effectors that mediate force-induced OPN up-
regulation have not previously been elucidated in hPLFs.
This study demonstrates for the first time that FAK is the
main downstream target of Rho/ROCK signaling in force-
exposed hPLFs, based on the following findings: (1) the
suppression of Rho and ROCK by specific inhibitors
attenuates FAK activation, and (2) transfecting cells with a
FAK ASO diminished OPN expression at both the mRNA
and protein levels in hPLFs that had been stimulated by
centrifugal force.
FAK is a major mechanosensitive kinase linked to inte-
grin signaling, and its role in mechanotransduction has been
widely studied using myocytes and nonmyocytes. Given
that FAK phosphorylation is correlated with the activation
of RhoA in most of the cell lines studied, it is not surprising
that centrifugal force would up-regulate OPN expression
through the cascade activation of Rho/ROCK/FAK path-
ways in hPLFs. There are several reports that detail the
different roles of OPN with regard to interactions between
FAK and integrins. Treating vascular smooth muscle cells
(VSMC) with OPN promoted migration of cells by inducing
FAK phosphorylation and integrin-linked kinase dephos-
phorylation [50]. OPN also increased integrin b3 expression
and induced a rapid and transient activation of FAK in
VSMCs [51]. These findings suggest that OPN acts as an
inducer of FAK/Rho kinase phosphorylation. More direct
evidence will be necessary to determine how mechanical
force and RhoA/ROCK signaling contribute to FAK phos-
phorylation and OPN induction in hPLFs.
Because mechanical stresses lead to cellular messages
and outcomes through similar mechanoreceptors and sig-
naling effectors in many types of cells and tissues, it has
been suggested that common signaling mechanisms are
involved in mechanotransduction pathways. In particular,
ERK1/2 is the most prominent kinase activated by
mechanical stimuli in most cells examined [36, 52, 53].
Many studies have shown that RhoA/ROCK and FAK are
downstream mechanosensors of integrins as well as
upstream effectors that induce ERK1/2 phosphorylation in
stress-exposed cells [52, 54, 55]. Similar to a previous
study [12, 37], our current findings demonstrate that ERK1/
2 is a central mediator of mechanical signals transmitted by
Rho/ROCK and FAK pathways in hPLFs.
ERK1/2 transmits mechanical signals into the nucleus
by activating transcription factors such as Elk1 and c-Fos
[38, 56, 57]. Elk1, one of the Ets family of transcription
factors, is a DNA-binding protein that activates transcrip-
tion through its phosphorylation [58]. According to a report
by El-Tanani et al. [59], the OPN promoter contains vari-
ous binding sites for transcriptional factors such as Ets,
TCF, and AP-1. Additionally, the binding of TCF/Elk1
123
Mol Cell Biochem (2010) 335:263–272
Fig. 8 Proposed model of the mechanotransduction pathways
involved in OPN up-regulation in centrifugal force-exposed hPLFs
protein to specific sites on the OPN promoter is necessary
to stimulate OPN expression mediated by ERK activation
[60]. This is supported by the finding that an ERK inhibitor
blocked Elk1 phosphorylation as well as OPN gene
expression stimulated by oscillatory fluid flow in human
osteoblast-like cells [38]. Our present results reveal that
when a centrifugal force is applied to hPLFs, the phos-
phorylation of Elk1 increases, and this increase is signifi-
cantly diminished by an ERK inhibitor but not by other
kinase inhibitors. In analogy to mechanical stimuli leading
to similar outcomes in other cells, the cascade phosphor-
ylation of ERK1/2 and Elk1 with attendant binding to TCF
and/or Ets domain appears to be, at least in part, associated
with OPN expression in force-exposed hPLFs. Further
studies are needed to clarify the relationship between ERK/
Elk1 and their target transcription factors in force-induced
up-regulation of OPN.
AP-1 is a dimeric transcription factor composed of
members of the Fos and Jun protein subfamilies. ERK
stimulates AP-1 activity by inducing c-Fos synthesis and
by enhancing c-Fos protein transactivation [56, 61]. Vari-
ous types of extracellular stress can up-regulate c-Fos in
diverse experimental systems [57, 62, 63]. Indeed,
mechanical stretching up-regulates c-Fos induction in
hPLFs [62], and our previous findings showed that cen-
trifugal force rapidly and transiently activates the DNA-
binding capacity of AP-1 through ERK and JNK-mediated
signaling in hPLFs. The current study shows that centrif-
ugal force-activated AP-1 DNA binding was significantly
attenuated by an ERK inhibitor in hPLFs. Therefore, we
suggest that the ERK/c-Fos pathway is in part related to the
force-induced up-regulation of OPN in hPLFs.
In summary, our present findings show that centrifugal
force up-regulates OPN expression at both the mRNA and
proteins levels in hPLFs, and that the applied force induces
Mol Cell Biochem (2010) 335:263–272
the rapid and transient phosphorylation of FAK, ERK1/2,
and Elk1. This study also reveals that inhibition of RhoA,
ROCK, or FAK by pharmacological inhibitors or by
transfecting cells with antisense oligonucleotides attenu-
ates the force-induced ERK phosphorylation and OPN
expression, where Elk1 and c-Fos act as downstream
effectors. Consequently, these findings demonstrate that
FAK signaling is critical to force-induced OPN up-regu-
lation in hPLFs and indicate that this event is mediated by
the Rho/ROCK and ERK-Elk1/ERK-c-Fos pathways as
upstream and downstream effectors of FAK, respectively,
as summarized in Fig. 8.
Acknowledgements This study was supported by a grant from the
Korea Healthcare Technology R&D Project, Ministry for Health,
Welfare & Family Affairs, Republic of Korea (A084283).
References
1. Sodek J, Limeback HF (1979) Comparison of the rates of syn-
thesis, conversion and maturation of type I and type III collagens
in rat periodontal tissues. J Biol Chem 254:10496–10502
2. Berkovitz BKB (1990) The structure of the periodontal ligament:
an update. Eur J Orthod 12:51–76
3. Lekic P, McCulloch CA (1996) Periodontal ligament cell popu-
lation: the central role of fibroblasts in creating a unique tissue.
Anat Rec 245:327–341
4. Kanzaki H, Chiba M, Shimizu Y, Mitani H (2001) Dual regula-
tion of osteoclast differentiation by periodontal ligament cells
through RANKL stimulation and OPG inhibition. J Dent Res
80:887–891
5. Kanzaki H, Chiba M, Shimizu Y, Mitani H (2002) Periodontal
ligament cells under mechanical stress induce osteoclastogenesis
by receptor activator of nuclear factor kappaB ligand up-regula-
tion via prostaglandin E2 synthesis. J Bone Miner Res 17:210–220
6. Wada N, Maeda H, Yoshimine Y, Akamine A (2004) Lipo-
polysaccharide stimulates expression of osteoprotegerin and
receptor activator of NF-kappaB ligand in periodontal ligament
fibroblasts through the induction of interleukin-1beta and tumor
necrosis factor-alpha. Bone 35:629–635
7. Gotoh Y, Salih E, Glimcher MJ, Gerstenfeld LC (1995) Char-
acterization of the major non-collagenous proteins of chicken
bone: identification of a novel 60 kDa non-collagenous phos-
phoprotein. Biochem Biophys Res Commun 208:863–870
8. Weinreb M, Shinar D, Rodan GA (1990) Different pattern of
alkaline phosphatase, osteopontin, and osteocalcin expression in
developing rat bone visualized by in situ hybridization. J Bone
Miner Res 5:831–842
9. Merry K, Dodds R, Littlewood A, Gowen M (1993) Expression
of osteopontin mRNA by osteoclasts and osteoblasts in modelling
adult human bone. J Cell Sci 104(Pt 4):1013–1020
10. Terai K, Takano-Yamamoto T, Ohba Y, Hiura K, Sugimoto M,
Sato M, Kawahata H, Inaguma N, Kitamura Y, Nomura S (1999)
Role of osteopontin in bone remodeling caused by mechanical
stress. J Bone Miner Res 14:839–849
11. Morinobu M, Ishijima M, Rittling SR, Tsuji K, Yamamoto H,
Nifuji A, Denhardt DT, Noda M (2003) Osteopontin expression in
osteoblasts and osteocytes during bone formation under mechan-
ical stress in the calvarial suture in vivo. J Bone Miner Res 18:
1706–1715
271
12. Jeon YM, Kook SH, Son YO, Kim EM, Park SS, Kim JG, Lee JC
(2009) Role of MAPK in mechanical force-induced up-regulation
of type I collagen and osteopontin in human gingival fibroblasts.
Mol Cell Biochem 320:45–52
13. Giachelli CM, Steitz S (2000) Osteopontin: a versatile regulator
of inflammation and biomineralization. Matrix Biol 19:615–622
14. Standal T, Borset M, Sundan A (2004) Role of osteopontin in
adhesion, migration, cell survival and bone remodeling. Exp
Oncol 26:179–184
15. Lao M, Marino V, Bartold PM (2006) Immunohistochemical
study of bone sialoprotein and osteopontin in healthy and dis-
eased root surfaces. J Periodontol 77:1665–1673
16. Lee A, Schneider G, Finkelstein M, Southard T (2004) Root
resorption: the possible role of extracellular matrix proteins. Am J
Orthod Dentofacial Orthop 126:173–177
17. Jimenez-Pellegrin C, Arana-Chavez VE (2007) Root resorption
repair in mandibular first premolars after rotation. A transmission
electron microscopy analysis combined with immunolabeling of
osteopontin. Am J Orthod Dentofacial Orthop 132:230–236
18. Christgau M, Caffesse RG, Schmalz G, D’Souza RN (2007)
Extracellular matrix expression and periodontal wound-healing
dynamics following guided tissue regeneration therapy in canine
furcation defects. J Clin Periodontol 34:691–708
19. Terashima Y, Shimabukuro Y, Terashima H, Ozasa M, Terakura
M, Ikezawa K, Hashikawa T, Takedachi M, Oohara H, Yamada
S, Murakami S (2008) Fibroblast growth factor-2 regulates
expression of osteopontin in periodontal ligament cells. J Cell
Physiol 216:640–650
20. Sharma CG, Pradeep AR (2006) Gingival crevicular fluid oste-
opontin levels in periodontal health and disease. J Periodontol
77:1674–1680
21. Sharma CG, Pradeep AR (2007) Plasma and crevicular fluid
osteopontin levels in periodontal health and disease. J Periodontal
Res 42:450–455
22. Wongkhantee S, Yongchaitrakul T, Pavasant P (2007) Mechan-
ical stress induces osteopontin expression in human periodontal
ligament cells through rho kinase. J Periodontol 78:1113–1119
23. Wongkhantee S, Yongchaitrakul T, Pavasant P (2008) Mechan-
ical stress induces osteopontin via ATP/P2Y1 in periodontal
cells. J Dent Res 87:564–568
24. Torsoni AS, Marin TM, Velloso LA, Franchini KG (2005) RhoA/
ROCK signaling is critical to FAK activation by cyclic stretch in cardiac
myocytes. Am J Physiol Heart Circ Physiol 289:H1488–H1496
25. Lal H, Verma SK, Smith M, Guleria RS, Lu G, Foster DM,
Dostal DE (2007) Stretch-induced MAP kinase activation in
cardiac myocytes: differential regulation through beta1-integrin
and focal adhesion kinase. J Mol Cell Cardiol 43:137–147
26. Albinsson S, Hellstrand P (2007) Integration of signal pathways
for stretch-dependent growth and differentiation in vascular
smooth muscle. Am J Physiol Cell Physiol 293:C772–C782
27. Chaturvedi LS, Marsh HM, Basson MD (2007) Src and focal
adhesion kinase mediate mechanical strain-induced proliferation
and ERK1/2 phosphorylation in human H441 pulmonary epi-
thelial cells. Am J Physiol Cell Physiol 292:C1701–C1713
28. Takeda H, Matozaki T, Fujioka Y, Takada T, Noguchi T, Yamao
T, Tsuda M, Ochi F, Fukunaga K, Narumiya S, Yamamoto T,
Kasuga M (1998) Lysophosphatidic acid-induced association of
SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family
kinase. Oncogene 16:3019–3027
29. Pirone DM, Liu WF, Ruiz SA, Gao L, Raghavan S, Lemmon CA,
Romer LH, Chen CS (2006) An inhibitory role for FAK in reg-
ulating proliferation: a link between limited adhesion and RhoA-
ROCK signaling. J Cell Biol 174:277–288
30. Zhang SJ, Truskey GA, Kraus WE (2007) Effect of cyclic stretch
on beta1D-integrin expression and activation of FAK and RhoA.
Am J Physiol Cell Physiol 292:C2057–C2069
123
272
31. Howard PS, Kucich U, Taliwal R, Korostoff JM (1998)
Mechanical forces alter extracellular matrix synthesis by human
periodontal ligament fibroblasts. J Periodontal Res 33:500–508
32. Redlich M, Palmon A, Zaks B, Geremi E, Rayzman S, Shoshan S
(1998) The effect of centrifugal force on the transcription levels
of collagen type I and collagenase in cultured canine gingival
fibroblasts. Arch Oral Biol 43:313–316
33. Redlich M, Roos H, Reichenberg E, Zaks B, Grosskop A, Bar
Kana I, Pitaru S, Palmon A (2004) The effect of centrifugal force
on mRNA levels of collagenase, collagen type-I, tissue inhibitors
of metalloproteinases and b-actin in cultured human periodontal
ligament fibroblasts. J Periodontal Res 39:27–32
34. Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248–254
35. Maulik N, Sato M, Price BD, Das DK (1998) An essential role of
NF-kappaB in tyrosine kinase signaling of p38 MAP kinase
regulation of myocardial adaptation to ischemia. FEBS Lett
429:365–369
36. Liedert A, Kaspar D, Blakytny R, Claes L, Ignatius A (2006)
Signal transduction pathways involved in mechanotransduction in
bone cells. Biochem Biophys Res Commun 349:1–5
37. Kook SH, Hwang JM, Park JS, Kim EM, Heo JS, Jeon YM, Lee
JC (2009) Mechanical force induces type I collagen expression in
human periodontal ligament fibroblasts through activation of
ERK/JNK and AP-1. J Cell Biochem 106:1060–1067
38. Wu CC, Li YS, Haga JH, Wang N, Lian IYZ, Su FC, Usami S,
Chien S (2006) Roles of MAP kinases in the regulation of bone
matrix gene expressions in human osteoblasts by oscillatory fluid
flow. J Cell Biochem 98:632–641
39. Franceschi RT, Xiao G, Jiang D, Gopalakrishnan R, Yang S,
Reith E (2003) Multiple signaling pathways converge on the
Cbfa1/Runx2 transcription factor to regulate osteoblast differen-
tiation. Connect Tissue Res 44(suppl 1):109–116
40. Xu C, Shen G, Yuan X, Kim JH, Gopalkrishnan A, Keum YS,
Nair S, Kong AN (2006) ERK and JNK signaling pathways are
involved in the regulation of activator protein 1 and cell death
elicited by three isothiocyanates in human prostate cancer PC-3
cells. Carcinogenesis 27:437–445
41. Katsumi A, Orr AW, Tzima E, Schwartz MA (2004) Integrins in
mechanotransduction. J Biol Chem 279:12001–12004
42. Schwartz MA, DeSimone DW (2008) Cell adhesion receptors in
mechanotransduction. Curr Opin Cell Biol 20:551–556
43. Cho KA, Ryu SJ, Oh YS, Park JH, Lee JW, Kim HP, Kim KT,
Jang IS, Park SC (2004) Morphological adjustment of senescent
cells by modulating caveolin-1 status. J Biol Chem 279:42270–
42278
44. Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N,
Matsuura Y, Kaibuchi K (1997) Formation of actin stress fibers
and focal adhesions enhanced by Rho-kinase. Science 275:1308–
1311
45. Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Zhu W,
Kadowaki T, Yazaki Y (1999) Rho family small G proteins play
critical roles in mechanical stress-induced hypertrophic responses
in cardiac myocytes. Circ Res 84:458–466
46. Makino A, Glogauer M, Bokoch GM, Chien S, Schmid-Schön-
bein GW (2005) Control of neutrophil pseudopods by fluid shear:
role of Rho family GTPases. Am J Physiol Cell Physiol 288:
C863–C871
47. Riento K, Ridley AJ (2003) Rocks: multifunctional kinases in cell
behaviour. Nat Rev Mol Cell Biol 4:446–456
123
Mol Cell Biochem (2010) 335:263–272
48. Yamashiro K, Myokai F, Hiratsuka K, Yamamoto T, Senoo K,
Arai H, Nishimura F, Abiko Y, Takashiba S (2007) Oligonu-
cleotide array analysis of cyclic tension-responsive genes in
human periodontal ligament fibroblasts. Int J Biochem Cell Biol
39:910–921
49. Chellaiah MA, Biswas RS, Rittling SR, Denhardt DT, Hruska KA
(2003) Rho-dependent Rho kinase activation increases CD44
surface expression and bone resorption in osteoclasts. J Biol
Chem 278:29086–29097
50. Li JJ, Han M, Wen JK, Li AY (2007) Osteopontin stimulates
vascular smooth muscle cell migration by inducing FAK phos-
phorylation and ILK dephosphorylation. Biochem Biophys Res
Commun 356:13–19
51. Han M, Wen JK, Zheng B, Liu Z, Chen Y (2007) Blockade of
integrin beta3-FAK signaling pathway activated by osteopontin
inhibits neointimal formation after balloon injury. Cardiovasc
Pathol 16:283–290
52. Whitmarsh AJ, Davis RJ (1996) Transcription factor AP-1 reg-
ulation by mitogen-activated protein kinase signal transduction
pathways. J Mol Med 74:589–607
53. Li C, Hu Y, Mayr M, Xu Q (1999) Cyclic strain stress-induced
mitogen-activated protein kinase (MAPK) phosphatase 1
expression in vascular smooth muscle cells is regulated by Ras/
Rac-MAPK pathways. J Biol Chem 274:25273–25280
54. Basdra ER, Papavassiliou AG, Huber LA (1995) Rab and rho
GTPases are involved in specific response of periodontal liga-
ment fibroblasts to mechanical stretching. Biochim Biophys Acta
1268:209–213
55. Hall A (2005) Rho GTPases and the control of cell behaviour.
Biochem Soc Trans 33(Pt 5):891–895
56. Davis RJ (2000) The JNK signal transduction pathway. Cell
103:239–252
57. Peake MA, Cooling LM, Magnay JL, Thomas PB, El Haj AJ
(2000) Selected contribution: regulatory pathways involved in
mechanical induction of c-fos gene expression in bone cells.
J Appl Physiol 89:2498–2507
58. Cruzalegui FH, Cano E, Treisman R (1999) ERK activation
induces phosphorylation of Elk-1 at multiple S/T-P motifs to high
stoichiometry. Oncogene 18:7948–7957
59. El-Tanani M, Platt-Higgins A, Rudland PS, Campbell FC (2004)
Ets gene PEA3 cooperates with beta-catenin-Lef-1 and c-Jun in
regulation of osteopontin transcription. J Biol Chem 279:20794–
20806
60. Denhardt DT, Mistretta D, Chambers AF, Krishna S, Porter JF,
Raghuram S, Rittling SR (2003) Transcriptional regulation of
osteopontin and the metastatic phenotype: evidence for a Ras-
activated enhancer in the human OPN promoter. Clin Exp
Metastasis 20:77–84
61. Yoshioka K, Deng T, Cavigelli M, Karin M (1995) Antitumor
promotion by phenolic antioxidants: inhibition of AP-1 activity
though induction of Fra expression. Proc Natl Acad Sci USA
92:4972–4976
62. Kletsas D, Basdra E, Papavassiliou AG (2002) Effect of protein
kinase inhibitors on the stretch-elicited c-Fos and c-Jun up-reg-
ulation in human PDL osteoblast-like cells. J Cell Physiol 190:
313–321
63. Li J, Chen G, Zheng L, Luo S, Zhao Z (2007) Osteoblast cyto-
skeletal modulation in response to compressive stress at physio-
logical levels. Mol Cell Biochem 304:45–52