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INTRODUCTION |
Mechanical loading applied to the skeleton is crucial to the
development and maintenance of bone integrity and architecture. A
decrease in the mechanical loading due to prolonged immobilization or
weightlessness in space reduces the bone formation rate, resulting in
bone loss (1-3). On the other hand, an increase in mechanical loading
causes a gain in bone density (4, 5). Thus, bone tissue is sensitive to
mechanical stimulation. Mechanical loading on bone generates
extracellular matrix deformation and fluid flow, and the mechanical
stimuli are translated to mechanical signals such as mechanical strain
and fluid shear stress, respectively (6). Evidence obtained from
in vitro studies indicates that osteocytes embedded in the
lacunae/canaliculi system and osteoblasts and bone cells lining the
bone surface are mechanosensors that detect load-derived mechanical
stimuli (7, 8). By these bone-forming cells, the mechanical stimuli are
translated into cellular signaling factors.
Mechanical stress induces the expressions of several kinds of proteins
in bone-forming cells such as insulin-like growth factor-I and -II,
transforming growth factor-
, osteocalcin, osteopontin, c-Fos,
nitric-oxide synthase, and cyclooxygenase-2
(COX-2,1 an isoform of
prostaglandin G/H synthase), as reported in previous studies (9-15).
In particular, the administration of NS-398, a selective inhibitor of
COX-2, and indomethacin to rats in vivo inhibited mechanical
loading-induced bone formation (16, 17), suggesting that prostaglandins
(PGs) are important mediators of the mechanical loading-induced bone
response. In addition, up-regulation of expression of some skeletal
growth factors including insulin-like growth factor-I and transforming
growth factor-
in response to mechanical stress was at least in part
mediated by the PG production (10, 18). PGs have anabolic effects on
proliferation and differentiation of bone-forming cells via diverse
signal transduction systems dependent on their concentration and
species (19-21). PGs also regulate the differentiation and function of
bone-resorbing cells such as osteoclasts (22, 23). Therefore, the
mechanical stress-induced PG production by bone forming-cells may
modulate the overall process of bone metabolism to adapt the skeleton
to the mechanical environment.
Production of PGs is kinetically controlled mainly by the release of
arachidonic acid and expression of COX-2 in response to a variety of
stimuli (24). Fluid shear stress has been reported to stimulate rapidly
PGE2 production in osteocytes through a cascade of
sequential activation of cytoskeleton-associated Ca2+
channel, phospholipase C, intracellular Ca2+, protein
kinase C (PKC), and phospholipase A2 (25). Regarding COX-2
induction, it has been reported that cox-2 expression
induced by fluid shear stress could be dependent on
cytoskeleton-integrin interactions and intracellular calcium release
mediated by inositol trisphosphate in osteoblastic MC3T3-E1 cells (26).
However, there has been no report indicating transcription factors or
transcriptional regulatory elements in the cox-2 promoter
region responsible for the shear stress-induced cox-2
transcription, whereas the cytokine- or growth
factor-dependent factors and regulatory elements have been
extensively reported (27-31).
In the present study, we attempted to identify transcription factors
and transcriptional regulatory elements located in 5'-flanking region
of the cox-2 promoter gene that contribute to the shear stress-induced cox-2 expression. Here, we report our
findings indicating that the cox-2 expression induced by
fluid shear stress was mediated by C/EBP
, AP-1, and CREB, which
bound to their respective sites on the cox-2 promoter gene
in osteoblastic MC3T3-E1 cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
MC3T3-E1 cells (2500 cells/cm2)
were seeded on type 1 collagen-coated slide glasses (Matsunami, Tokyo,
Japan; 25 × 50 × 1 mm) in 100-mm plastic dishes and were
cultured in
-minimum essential medium (
-MEM, ICN Biomedicals
Inc., Aurora, OH) containing 10% fetal bovine serum (FBS, Intergen,
NY) and 100 units/ml of penicillin G at 37 °C in a humidified
CO2 incubator (5% CO2, 95% air) as described
previously (21). After 4 days, the culture medium was removed and
replaced with fresh
-MEM containing 10% FBS. The removed medium was
stored at 4 °C to be used as conditioned medium. The cells were
cultured further for 3 more days prior to use in fluid shear stress experiments.
Shear Stress Experiments--
A single-pass flow-through system
was used. After MC3T3-E1 cells had been cultured on a slide glass (1-mm
thick), the slide glass was carefully removed from and mounted on a
parallel plate flow chamber that had been constructed by sandwiching
silicone gaskets (1.5, 2, or 3 mm thick) between two acrylic plates,
creating a flow channel (0.5, 1, or 2 mm deep × 25 mm wide ×50
mm long). Then the cells in the chamber were exposed to the fluid shear stress, which was generated by circulating the conditioned medium (0.36 ml/s) through a hydrostatic pump connected to the reservoirs at
37 °C in a CO2 incubator. The pH of the medium was kept
constant by gassing with humidified 95% air and 5% CO2.
As a control, the cells in the flow chamber were incubated for the same
duration without having been exposed to the shear stress. When the rate of fluid flow is constant in the chamber, the magnitude of the shear
stress is inversely proportional to the square of the depth of the flow
channel. When the flow rate of the conditioned medium was 0.36 ml/s,
the shear stress at 0.5-, 1.0-, and 2.0-mm depths of the flow channel
was calculated to be 2.88, 0.72, and 0.18 dynes/cm2, respectively.
Reverse Transcription-Polymerase Chain Reaction (PCR)--
After
MC3T3-E1 cells had been subjected to shear stress for the desired time,
total RNA (1 µg) extracted from the cells was used as a template for
cDNA synthesis. cDNA was prepared by use of a Superscript II
preamplification system (Life Technologies, Inc.). Primers were
synthesized on the basis of the reported mouse cDNA sequences for
COX-2 and
-actin. Sequences of the primers used for PCR were as
follows: cox-2 forward, 5'-GGG TTG CTG GGG GAA GAA ATG
TG-3'; COX-2 reverse, 5'-GGT GGC TGT TTT GGT AGG CTG TG-3';
-actin
forward, TCA CCC ACA CTG TGC CCA TCT AC-3';
-actin reverse, 5'-GAG
TAC TTG CGC TCA GGA GGA GC-3'. Amplification was carried out for 22-27
cycles under saturation, each at 94 °C, 45 s; 60 °C, 45 s; 72 °C, 1 min in a 50-µl reaction mixture containing 0.5 µl of
each cDNA, 50 pmol of each primer, 0.2 mM dNTP, and 1.25 units of Taq DNA polymerase (Qiagen, Inc., Valencia, CA). After amplification, 10 µl of each reaction mixture was analyzed by
1.5% agarose gel electrophoresis, and the bands were then visualized by ethidium bromide staining. The PCR products for cox-2 and
-actin were 479 and 538 bp, respectively.
Antibodies--
Anti-COX-2, anti-c-Jun (sc-44x), anti-c-Fos
(sc-253x), and anti-C/EBP
(sc-746x) antibodies were purchased from
Santa Cruz Biotechnology (San Diego, CA). Anti-CREB and
anti-phospho-CREB antibodies were purchased from Upstate Biotechnology,
Inc. (Lake Placid, NY).
Western Blot Analysis--
After exposure to shear stress,
MC3T3-E1 was washed with PBS, scraped into a solution consisting of 10 mM sodium phosphate (pH 7.5), 150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM
EDTA, 1 mM p-aminoethylbenzenesulfonyl fluoride (p-ABSF), 10 µg/ml leupeptin, and 10 µg/ml aprotinin,
and sonicated for 15 s. The protein concentration in the cell
lysate was measured with a bicinchoninic acid protein assay kit
(Pierce). Samples containing equal amounts of protein were subjected to
10% SDS-polyacrylamide gel electrophoresis (PAGE), and the proteins
separated in the gel were subsequently electrotransferred onto a
polyvinylidene difluoride membrane. After having been blocked with 5%
skim milk, the membrane was incubated with anti-COX-2 antibody or
nonimmune rabbit IgG and subsequently with peroxidase-conjugated
anti-rabbit IgG antibody. Immunoreactive proteins were visualized with
Western blot chemiluminescence reagents (PerkinElmer Life Sciences)
following the manufacturer's instructions.
Preparation of the 5'-flanking Region of Mouse cox-2 Gene and
Construction of Luciferase Reporter Vectors--
Luciferase reporter
pGL2 plasmid including 5'-flanking region of the murine
cox-2 gene from
3195 to +39 bp was kindly provided by Dr.
David L. DeWitt (Michigan State University). DNA fragments of various
lengths of the cox-2 promoter regions were prepared by PCR
using the above plasmid as a template and PyrobestTM DNA
polymerase (Takara, Kyoto, Japan). Mutated fragments were prepared by
two-stage bridge PCR, using mutated primers previously reported by
Brunner et al. (32). These fragments were inserted into pGL2
basic vectors (Promega, Madison, WI), by using a Ligation kit version
II® (Takara).
Transfection of MC3T3-E1 Cells with Plasmids and Luciferase
Assay--
MC3T3-E1 cells were cultured for 4 days on type 1 collagen-coated slide glasses in 100-mm dishes containing
-MEM
supplemented with 10% FBS. For transfection, the cells were treated
for 24 h with plasmid DNA (0.7 µg) containing cox-2
promoter and luciferase reporter gene, standard plasmid DNA (0.4 µg)
containing
-galactosidase gene, and 18 µl of Effectene
transfection reagent® (Qiagen) in 4 ml of
-MEM with 10% FBS. Then
the medium was changed to 15 ml of
-MEM with 10% FBS, and the
transfected cells were further cultured for 3 days. Thereafter, the
transfected cells were placed in the flow shear stress chamber and
subjected to fluid shear stress (2.88 dynes/cm2) for 6 h at 37 °C. Control cells were also placed in other chambers without
fluid flow for the same period. The cells were then washed twice with
cold PBS and were scraped in 200 µl of Reporter lysis buffer
(Promega). Luciferase activities in the cell lysate were measured by
using a microplate luminometer (MicroLumat LB96P, EG & G Berthold,
Aliquippa, PA) and a luciferase assay system (Promega), according to
the manufacturer's instruction. For determination of
-galactosidase
activity, the cell lysate (15 µl) was incubated in a 335-µl
reaction buffer (0.1 M sodium phosphate (pH 7.5, 2 mM o-nitrophenyl-
-galactopyranoside), 10 mM KCl, 1 mM MgCl2, 0.1% Triton
X-100, 5 mM
-mercaptoethanol) at 37 °C for 24 h. The reaction was then terminated with 150 µl of 1 M
sodium carbonate, and the absorbance was measured at 420 nm. The
luciferase activities were normalized on the basis of
-galactosidase
activities. A part of the cell lysate was used to fluorometrically
determine DNA content by the method of Kissane and Robins (33).
Data Analysis of Luciferase Activity--
In individual
luciferase assays for deletion or mutation analysis, we always provided
control cells that had been transfected with a reporter plasmid
containing the cox-2 promoter region from
959 to +39 bp.
The control cells were also subjected to shear stress together with
cells transfected with other deleted or mutated reporter plasmids. The
normalized luciferase activities in the cells transfected with various
reporter plasmids were presented as percentage values, compared with
the value of luciferase activities of the shear stress-loaded cells
that had been transfected with the reporter plasmid containing the
cox-2 promoter region from
959 to +39 bp.
Preparation of Nuclear Extract--
After MC3T3-E1 cells had
been subjected to fluid shear stress for times indicated in the
legends, the cells were washed twice with ice-cold PBS, incubated for
10 min on ice in 1 ml of ice-cold buffer A (10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1 mM p-ABSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin), and
scraped. The cell lysates were incubated further for 10 min on ice and
then transferred to tubes. The nuclei obtained by centrifugation for 1 min at 5,000 × g were extracted by a 30-min incubation
in ice-cold buffer C, consisting of 50 mM Hepes (pH 7.5),
420 mM KCl, 0.1 mM EDTA, 5 mM
MgCl2, 20% glycerol, 1 mM dithiothreitol, 1 mM p-ABSF, 2 µg/ml aprotinin, 2 µg/ml
pepstatin, 2 µg/ml leupeptin. The extracts then were centrifuged at
14,000 × g for 30 min, and the supernatants were used
for the electrophoretic mobility shift assay (EMSA).
EMSA--
As shown in Table I, three oligonucleotides were
synthesized on the basis of the sequence of putative binding sites of
C/EBP-
, AP-1, and CREB located in the promoter region of the murine
cox-2 gene. The AP-1 probes and CREB probes were partially
mutated to avoid the cross-bindings of other transcription factors,
because the sequences of probes for AP-1 and CREB overlapped in part
with CRE-binding site and AP-1- and E-box-binding sites, respectively. The oligonucleotides were annealed with their complementary
oligonucleotides. The double-stranded oligonucleotides were end-labeled
with [
-32P]ATP by using T4 polynucleotide kinase
(Promega) according to the manufacturer's instructions and were used
as probes for EMSA. The nuclear extracts (1.8 µg of protein) were
incubated in binding buffer (10 mM Tris-HCl (pH 7.5), 4%
glycerol, 50 mM NaCl, 1 mM MgCl2,
0.5 mM EDTA, 0.5 mM dithiothreitol containing
32P-labeled C/EBP
, AP-1, and CREB probes) for 20 min at
room temperature. The protein-DNA complexes were resolved by PAGE (5%
gel) in 0.5× TBE buffer and visualized by autoradiography. For
supershift experiments, the nuclear extracts were incubated with
anti-c-Jun, anti-c-Fos, anti-C/EBP-
, anti-CREB, or anti-phospho-CREB
antibody for 30 min on ice after binding to the oligonucleotides and
then were subjected to PAGE.
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RESULTS |
Up-regulation of COX-2 Expression in Osteoblastic MC3T3-E1 Cells by
Fluid Shear Stress--
When osteoblastic MC3T3-E1 cells were exposed
to 2.88 dynes/cm2 of fluid shear stress, expression of
cox-2 mRNA was increased as early as 1 h after the
start of exposure and reached a maximum at 3 h (Fig.
1A). The increase in the
expression continued at least for 9 h. The up-regulation of
cox-2 mRNA expression depended on the magnitude of the
fluid shear stress, with a significant increase even at 0.18 dynes/cm2 (Fig. 1B). In addition, the fluid
shear stress also induced the expression of COX-2 protein in a
time-dependent manner, with a maximal effect at 6 h
after the shear stress application (Fig. 1C).

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Fig. 1.
Effects of shear stress on COX-2 expression
in MC3T3-E1 cells. A, osteoblastic MC3T3-E1 cells were
exposed (S) or not exposed (N) to fluid shear
stress (2.88 dynes/cm2) for the indicated times. Then total
RNA was extracted, and expression of COX-2 and -actin mRNA in
the cells was analyzed by reverse transcription-PCR. Numbers
in parentheses indicate the number of PCR cycles.
B, the cells were subjected to various magnitudes of the
fluid shear stress for 1 h. Numbers in
parentheses indicate the number of PCR cycles. C,
after the cells were exposed (S) or not exposed
(N) to the shear stress (2.88 dynes/cm2) for the
indicated times, the cells were extracted. The cell extracts were
subjected to Western blotting analysis with anti-COX-2 antibody.
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Functional Activity of cox-2 Promoter in Response to Fluid Shear
Stress--
About 1,000 bp of the 5'-flanking region of the mouse
cox-2 gene contained various putative transcription response
elements as described previously (28). When MC3T3-E1 cells were
transfected with luciferase-reporter plasmid including the 5'-flanking
region of the cox-2 gene (
959 to +39 bp), luciferase
activities in the cells were time-dependently increased in
response to the fluid shear stress (2.88 dynes/cm2, Fig.
2). A maximal increase was observed at
6 h after the shear stress application, consistent with the
induction of COX-2 protein determined by the Western blotting analysis
(Fig. 1C). The increase in the luciferase activity was
maintained up to 9 h.

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Fig. 2.
Fluid shear stress-induced COX-2 protein and
luciferase activity of WT COX-2 reporter plasmids in osteoblastic
MC3T3-E1 cells. Osteoblastic MC3T3-E1 cells were transiently
transfected with 0.7 µg of pGL2 reporter plasmid including the COX-2
gene promoter from 959 to +39 bp (WT [-959]) and 0.4 µg of
-galactosidase plasmid. The transfected cells were subjected or not
subjected to the shear stress (2.88 dynes/cm2) for the
indicated times. The cells were then extracted in a lysis buffer, and
the extracts were used to determine the activities of luciferase and
-galactosidase. The luciferase activity in the transfectants was
normalized to the -galactosidase activity and then revised by the
intracellular DNA content.
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To identify what regions of the cox-2 promoter contributed
to the shear stress-induced cox-2 transcription, we next
constructed eight luciferase reporter plasmids containing various
lengths of 5'-flanking region of the murine cox-2 gene as
shown in Fig. 3. After each plasmid
vector was introduced to MC3T3-E1 cells, the luciferase activities were
measured at 6 h after application of fluid shear stress (2.88 dynes/cm2). As shown in Fig. 3, in the cells transfected
with a plasmid having a region of cox-2 gene promoter from
959 to + 39 bp, an ~4-fold increase in the activity was induced by
the fluid shear stress. We defined this luciferase activity as 100%
for comparison to the activities in the shear stress-loaded or
-unloaded cells transfected with plasmids containing various deleted
regions of the cox-2 gene promoter. The shear stress-induced
luciferase activities were not decreased but rather increased by
transfection with plasmids deleted from
959 to
172 bp. However,
when the region from
172 to
100 bp in the promoter was deleted, the
stimulatory effect of the shear stress on luciferase activity decreased
markedly (Fig. 3). The shear stress-induced luciferase activity in the cells transfected with the plasmid deleted from
100 to
79 bp was
the same as that in the cells transfected with the plasmid deleted from
172 to
100 bp. In addition, deletions from
79 to
46 bp further
reduced the shear stress-induced luciferase activity. These deletion
data suggested that two regions (
172 to
100 bp and
79 to
46 bp)
represented possible shear stress-response elements. Based on analysis
of consensus sequence, these regions contained presumed
cis-elements for C/EBP
, AP-1, CRE, and E-box. To
eliminate the possibility that the decreases in shear stress-induced luciferase activity were attributable to the difference in the length
of the promoter region in the plasmids used, we introduced mutations in
the presumed response elements in the same length of cox-2
promoter (
959 to +39 bp) gene, as shown in Fig.
4. Introduction of a mutation into CRE
(1) (
447 to
440 bp), NF-
B (
401 to
393 bp), C/EBP
(2)
(
93 to
85 bp), and E-box (
53 to
48 bp) did not affect the
stimulation of luciferase activity by the fluid shear stress. On the
other hand, a mutations in C/EBP
(1) (
138 to
130 bp), AP-1
(
73 to
61 bp), or CRE (2) (
59 to
52 bp) reduced the shear
stress-induced luciferase activities to 41, 52, and 34%, respectively,
of the value for the wild-type reporter, suggesting these sites to be
shear stress-response elements. Furthermore, to confirm the functional
elements responsive to shear stress, we performed double and triple
mutation analyses (Fig. 5). When both
C/EBP
(1) (
138 to
130 bp) and CRE (2) (
59 to
52 bp) sites
were mutated, the shear stress-induced luciferase activity was further
decreased to 14% of that in the shear stress-loaded cells transfected
with the wild-type reporter plasmid containing the cox-2
promoter gene (
959 to +39 bp). In addition, by triple mutation in
C/EBP
(1) (
138 to
130 bp), AP-1 (
73 to
61 bp), and CRE (2)
(
59 to
52 bp) sites, the stimulation of luciferase activity in
response to the fluid shear stress fell to the level of the pGL2 basic
vector. These results suggested that C/EBP
, AP-1, and CREB acted as
functional transcription factors for up-regulation of cox-2
transcript expression in response to the fluid shear stress.

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Fig. 3.
Construction of luciferase reporter plasmids
containing COX-2 gene promoter regions and deletion analysis of COX-2
gene promoters. Putative consensus sequences in the 5'-flanking
region of murine COX-2 gene are illustrated in the upper
left. Each deleted promoter fragment was inserted into a pGL2
basic luciferase vector. Numbers indicate distance in bp
from the start site of transcription. Osteoblastic MC3T3-E1 cells were
transiently transfected with these plasmids along with the
-galactosidase plasmid. The transfected cells were exposed or not
exposed to shear stress at 2.88 dynes/cm2 for 6 h, and
then the activities of luciferase and -galactosidase were measured.
The luciferase activities were normalized to the -galactosidase
activities. The normalized luciferase activities in the cells
transfected with various reporter plasmids are presented as percentage
values, compared with the value of luciferase activities of the shear
stress-loaded cells that had been transfected with the reporter plasmid
containing the COX-2 promoter region from 959 to +39 bp (WT
[ 959]).
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Fig. 4.
Site mutation analysis of luciferase
activities in response to fluid shear stress. The COX-2 gene
promoter ( 959 to +39 bp) was mutated at each putative transcriptional
regulatory element by the two-stage Bridge PCR method. The mutated
COX-2 gene promoters were ligated into pGL2 basic luciferase vectors.
Lowercase letters in the upper sequence of each
promoter indicate mutated bases, and the lower sequence
shows wild-type bases. Osteoblastic MC3T3-E1 cells were transiently
transfected with the wild-type ( 959 to +39 bp) and mutated construct
along with the -galactosidase plasmid. The transfected cells were
subjected or not subjected to shear stress at 2.88 dynes/cm2 for 6 h and then assayed for luciferase and
-galactosidase activities. The luciferase activities were normalized
to the -galactosidase activities. The normalized luciferase
activities in the cells transfected with various reporter plasmids are
presented as percentage values, compared with the value of luciferase
activities of the shear stress-loaded cells that had been transfected
with the reporter plasmid containing the COX-2 promoter region from
959 to +39 bp (WT [ 959]).
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Fig. 5.
Double and triple mutation analyses of
luciferase activities in response to fluid shear stress. C/EBP
( 138 to 130 bp), AP-1 ( 73 to 61 bp), and CRE (2) ( 59 to
52 bp) sites were mutated (cross) by the two-stage Bridge
PCR method. The double- and triple-mutated COX-2 gene promoters were
ligated into pGL2 basic luciferase vectors. The MC3T3-E1 cells were
transiently transfected with the wild-type and mutated constructs. The
transfected cells were subjected or not subjected to shear stress for
6 h at 2.88 dynes/cm2 and then the activities of
luciferase and -galactosidase were measured. The luciferase
activities were normalized to the -galactosidase activities. The
normalized luciferase activities in the cells transfected with various
reporter plasmids are presented as percentage values, compared with the
value of luciferase activities of the shear stress-loaded cells that
had been transfected with the reporter plasmid containing the COX-2
promoter region from 959 to +39 bp (WT [ 959]).
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Electrophoretic Mobility Shift Assay Targeting Possible Shear
Stress-response Elements--
To identify further the possible shear
stress-response elements, we carried out an EMSA using the nuclear
extracts from MC3T3-E1 subjected to fluid shear stress. As shown in
Table I, we constructed three
double-stranded oligonucleotide probes for C/EBP
, AP-1, and CREB
based on the sequence of each binding site in the cox-2 promoter gene. When the C/EBP
probe was incubated with nuclear extracts from shear stress-unloaded cells, the band indicating their
complex was observed in the gel (Fig.
6A). The formation of the
complex increased with fluid shear stress loading, the increase being
significant as early as 1 h after the application of the shear
stress. When the probe and the nuclear extracts were incubated with
anti-C/EBP
antibody, the complex was further shifted to the upper
position in the gel, whereas other antibodies did not change the
mobility of the complex. Likewise, the fluid shear stress increased the
formation of the complex with the nuclear extracts and AP-1 probe, and
the complex was supershifted by incubating with anti-AP-1 antibody but
not with anti-CREB and anti-C/EBP
antibodies (Fig. 6B).
These results suggest that the shear stress increased the bindings of
C/EBP
and AP-1 to their respective sites in cox-2
promoter in MC3T3-E1 cells. On the other hand, the fluid shear stress
did not affect the binding of the nuclear extracts to the CREB probe,
whereas the shifted bands were recognized by anti-CREB antibody but not
by anti-AP-1 antibody (Fig. 6C). However, it was reported
that a translocation of CREB to the nucleus or its binding to DNA was
independent of activation of transcription of target genes and that the
phosphorylation of CREB positively regulated the activation (34).
Therefore, by the supershift experiments with phospho-CREB antibody, we
finally examined the effect of fluid shear stress on the
phosphorylation of CREB in the complex of the nuclear extracts with
CREB probe at 3 h. We found that the fluid shear stress at least
in part induced the phosphorylation of CREB in their complex,
suggesting the transactivation of CREB in the cells.
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Table I
Syntheses of double-stranded oligonucleotides as probes for mouse
cox-2 gene
The constructed three double-strand oligonucleotide probes for C/EBP
, AP-1, and CREB were synthesized based on the sequence of each
binding site in mouse cox-2 promoter gene. Sequences in
boxes indicate putative binding site for C/EBP , AP-1, or CREB,
respectively. Lowercase letters show mutated bases. The AP-1 probes and
CREB probes were partially mutated to avoid the cross-bindings of other
transcription factors, because the sequences of probes for AP-1 and
CREB overlapped in part with CRE-binding site and AP-1- and E-
box-binding sites, respectively, indicated by underlines.
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Fig. 6.
Electrophoretic mobility shift assay
targeting C/EBP , AP-1, and CRE sites.
Osteoblastic MC3T3-E1 cells were exposed (S) or not exposed (N) to
fluid shear stress (2.88 dynes/cm2) for 1 or 3 h.
After the exposure, nuclear extracts were prepared. The extracts (1.8 µg) were incubated with 32P-labeled oligonucleotide
probes for C/EBP (A), AP-1 (B), or CRE
(C) site in the presence or absence of normal IgG,
anti-C/EBP , anti-c-Jun/AP-1, anti-c-Fos, anti-CREB or
anti-phospho-CREB antibody, and the mixtures were then subjected to
PAGE (5% gel). Closed and open arrows indicate
shifted and supershifted bands, respectively; and dotted
arrow in C shows supershifted band with
anti-phospho-CREB antibody.
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DISCUSSION |
Mechanical loading on bone generates extracellular matrix
deformation and fluid flow, and the mechanical stimuli are translated into mechanical signals such as mechanical strain and fluid shear stress, respectively. These mechanical signals could adaptively change
functions of bone-forming cells (osteocytes and osteoblasts). The
results of extensive in vivo and in vitro studies
indicated that these mechanical signals induced the expression of
COX-2, an inducible isoform of prostaglandin G/H synthase, via a
complexity of signal transduction systems (15, 26, 35-37). However,
little is known about the mechanism that regulates cox-2
transcription in response to mechanical stress. In this study, we
demonstrated that the cox-2 expression induced by fluid
shear stress was mediated by C/EBP
, AP-1, and CREB, which bound to
their respective elements on the cox-2 gene promoter in
osteoblastic MC3T3-E1 cells. To our knowledge, this is the first report
defining the shear stress-response elements in the cox-2
promoter in bone-forming osteoblasts.
The types of mechanical stimulus that produce the adaptive response of
bone-forming cells are still under investigation in in vivo
studies. Evidence obtained from in vitro study has, however, shown that bone-forming cells are more sensitive to fluid shear stress
than to mechanical strain, suggesting that the fluid shear stress could
be a dominant signal in mechanotransduction (38, 39). When mechanical
loading is applied to bone, the flow of extracellular fluid filled in
the space between canaliculi and osteocyte process is increased by the
local deformation. A magnitude of 8-30 dynes/cm2 of fluid
shear stress on the osteocyte process is predicted during physiological
mechanical loading (40). Although osteocytes are considered to be
primary cells responsible for mechanosensing (8), the fluid shear
stress even at a low magnitude of the shear stress (<1
dynes/cm2) also increased the cox-2 mRNA
expression in osteoblastic MC3T3-E1, as demonstrated in this study.
These data suggest that osteoblasts as well as osteocytes are sensitive
to mechanical loading beyond expectation.
In this study, we attempted to identify transcription response elements
in the cox-2 promoter region that contribute to the induction of cox-2 mRNA expression in response to the
fluid shear stress. In the 5'-flanking region from
959 to +39 bp of
COX-2 gene promoter, there are two CRE elements (CRE (1) (
447 to
440 bp), CRE (2) (
59 to
52 bp)), two C/EBP
elements (C/EBP
(1) (
138 to
130 bp), C/EBP
(2) (
93 to
85 bp)), NF-
B element (
401 to
393 bp), AP-1 element (
73 to
61 bp), E-box (
53 to
48 bp), and TATA-box (
30 to 25 bp). The deletion analysis indicated that the shear stress-induced luciferase responses were dependent on two regions, from
172 to
100 bp and from
79 to
46
bp, which include C/EBP
(1), AP-1, or CRE (2) sites, and E-box. The
single mutation of the each site of C/EBP
(1), AP-1, and CRE (2),
but not that of the E-box, decreased the shear stress-induced
luciferase activities. In addition, the triple mutations of C/EBP
(1), AP-1, and CRE sites abolished the shear stress-induced luciferase
response. These results indicate that C/EBP
(1), AP-1, or CRE (2)
sites are required for the induction of COX-2 mRNA
expression by the fluid shear stress.
The involvement of these sites in the shear stress-induced COX-2
mRNA expression was also confirmed by the mobility gel shift assays. The bindings of C/EBP
and AP-1 to their respective
oligonucleotides were increased by exposure to the fluid shear stress,
suggesting a transactivation by these transcription factors. Such
transactivation has been demonstrated to require phosphorylation of the
factors (41-44). Although the CREB binding to its CRE site was
unaffected by the shear stress, phosphorylation of CREB was induced by
the shear stress. The activated function of CREB is also modulated by
phosphorylation by several kinases (32, 44) and is mediated by
coactivators such as CBP and p300 (45). Thus, shear stress-induced kinase activities could have induced the phosphorylation of the above
transcription factors, by which the COX-2 transcription could be
activated. Several signal transduction pathways for response to fluid
shear stress have been proposed. In bone and endothelial cells, the
fluid shear stress induced activation of a variety of protein kinases
and phospholipases (PLs), suggesting that putative mediators for the
mechanotransduction are Ca2+ channels, phospholipase C,
protein kinase (PK) A, PKC, phospholipase A2, protein
tyrosine kinases, CaMK-II, or mitogen-activated protein kinases (15,
25, 26, 46-48). Both C/EBP
and CREB are known to be phosphorylated
by PKA, PKC, CaMK-II or p38 mitogen-activated protein kinase (49-51),
and AP-1 is phosphorylated by c-Jun N-terminal kinase and p42/44
mitogen-activated protein kinase (52). It remains to be clarified which
signal transduction pathways dominantly contribute to the induction of
COX-2 expression during shear stress loading.
C/EBP
, AP-1, and CREB have a leucine zipper domain for
dimerization. It was reported that c-Jun and CREB formed a
heterocomplex, and this complex played an important role in the
transcription of some genes (53-55). In addition, evidence concerning
cross-talk in signal transduction demonstrated that AP-1 efficiently
transactivates CRE sequences and that Fos and Jun efficiently bind and
cooperate in activating CRE promoter elements (56). It is therefore
possible that these transcription factors associate with each other and regulate the COX-2 transcription in combination in response to fluid
shear stress. In this study, however, only antibody against a given
factor could recognize the shifted band of this factor with its
respective oligonucleotide. Thus, these transcription factors seem to
individually regulate the COX-2 transcription.
Besides sites of C/EBP
(
138 to
130 bp), AP-1 (
73 to
61 bp),
and CRE (
59 to
52 bp), other putative response elements exist in
the promoter region of COX-2 gene, another CRE site (
447 to
440
bp), NF-
B site (
401 to
393 bp), and another C/EBP
(
93 to
85 bp). However, the deletion and mutation of these elements had no
effect on the fluid shear stress-induced increase in luciferase activity, suggesting that these sites are not associated with the shear
stress-activated COX-2 transcription. In addition, the COX-2 promoter
region also contains a sequence (GAGACC,
305 to
300 bp) of shear
stress response element (SSRE), which was initially reported in the
promoter of the PDGF-B gene (57). However, the deletion of
the COX-2 promoter region from
366 to
172 bp including the SSRE
site failed to decrease the shear stress-induced luciferase response,
implying the independence of the response from SSRE. On the contrary,
the deletion (
366 to
172 bp) rather enhanced the luciferase
response, suggesting that there might be suppressor sites in this region.
Regarding NF-
B, evidence has been accumulating that shear stress
regulates gene expression of endothelial cells via activation of
NF-
B (58). Bhullar et al. (59) reported that shear
stress-induced NF-
B translocation into the nucleus was dependent on
integrin associated with cytoskeleton in endothelial cells. Pavalko
et al. (15) reported that an inhibition of the cytoskeleton
organization reduced COX-2 or c-Fos expression induced by 12 dynes/cm2 of shear stress in MC3T3-E1 cells; however, the
magnitude of the shear stress was about 5 times higher than that in
this study. Taken together, these results suggest that
mechanotransduction requires cytoskeleton-integrin interactions and
raise the possibility that NF-
B may be involved in the activation of
COX-2 transcription by strong shear stress. In addition, the activation
of NF-
B has been reported to trigger the COX-2 expression elicited
by some cytokines such as IL-1
and TNF-
in a variety of cells
(60, 61). Yamamoto et al. (28) demonstrated that the same
mutated sequences of NF-
B site employed in this study inhibited both the NF-
B DNA binding and the NF-
B-dependent COX-2
transcription induced by TNF-
in MC3T3-E1 cells. In our present
study, however, the mutation and deletion of the NF-
B site did not
affect the increase in luciferase activity induced by shear stress.
Thus, transcription response elements for COX-2 gene seem to vary in the types of stimuli and cells, but it should be noted that NF-
B might regulate the shear stress-induced COX-2 induction under a high
magnitude of shear stress.
In conclusion, our present study demonstrates that C/EBP
, AP-1, and
CREB sites located at
138 to
130 bp,
73 to
61 bp, and
59 to
52 bp, respectively, in a COX-2 promoter region were required for the
COX-2 transcription induced by fluid shear stress and that the shear
stress increased the DNA binding activity of C/EBP
and AP-1 and
enhanced CREB phosphorylation. These data suggest that C/EBP
, AP-1,
and CREB via each DNA-binding site regulate the fluid shear
stress-induced COX-2 expression.