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INTRODUCTION |
Skeletal systems are maintained by continuous bone remodeling.
Mechanical loading, as well as a number of biochemical factors, regulates this bone remodeling. Mechanotransduction in bone has been
proposed to involve a variety of biophysical signals including electrical potentials (streaming potentials and piezoelectric effects)
and direct transduction of matrix strain. Recent studies suggest that
shear stress is an important biophysical signal in bone cell
mechanotransduction (1-3). Indeed, experiments designed to
discriminate between flow and strain effects suggest that fluid flow-induced shear stress is a more potent stimulator of bone cells
than substrate deformation (4, 5). As bone tissue is loaded in
vivo, extracellular fluid in the canalicular network experiences a
heterogeneous pressurization in response to the deformation of the
mineralized bone matrix, resulting in generation of fluid flow along
pressure gradients. When loading is removed, pressure gradients and
flows are reversed. These fluid motions are dynamic and oscillatory in
nature. Recently, Jacobs et al. (6) demonstrated that
oscillating fluid flow
(OFF),1 similar to what a
bone cell might experience in vivo, mobilizes cytosolic
calcium in osteoblastic cells. This was the first study to examine the
effect of OFF on bone cells. Other studies have demonstrated that
steady or pulsating fluid flow regulates many biochemical factors such
as cytosolic calcium (4, 6), cAMP (1), prostaglandin E2 (2), inositol
trisphosphate (2), nitric oxide (7), cyclooxygenase-2 mRNA (8, 9),
and c-Fos (9) in osteoblastic cells. However, the precise mechanism by which bone cells convert biophysical signals, such as fluid
flow-induced shear stress, into these biochemical signals remains unclear.
A number of paracrine and autocrine factors have been identified that
control bone remodeling. Tumor necrosis factor (TNF)-
, a cytokine
synthesized in the bone microenvironment, has been shown to exert
pleiotropic effects on osteoblasts and osteoblast-like cells (10, 11).
It has also been shown that TNF-
increases the production of
interleukin-6 and macrophage colony-stimulating factor in
osteoblastic cells, thereby indirectly promoting differentiation of
osteoclasts and enhancing bone resorption (12, 13). In addition, the
production of TNF-
in pathological conditions such as estrogen
deficiency and rheumatoid arthritis has been suggested to result in
osteopenia and bone destruction adjacent to areas of inflammation
(14-16).
We previously demonstrated that TNF-
induces the expression of
intracellular adhesion molecule (ICAM)-1 through transcription factor
NF-
B activation in osteoblasts, leading to the promotion of bone
resorption (17, 18). The transcription factor NF-
B was first
identified as a protein that binds to a specific DNA site in the
intrinsic enhancer of the Ig
light chain gene. It is composed of
homo- or heterodimers of members of the Rel family that control the
expression of numerous genes involved in the immune and inflammatory
responses, cell adhesion, and growth control. Moreover, NF-
B plays a
role as a primary regulator of the stress response. NF-
B can be
rapidly activated by many types of extracellular stimuli, including
viral infection, bacterial products, oxidative stress, and physical
stress (for reviews, see Refs. 19-21). Additionally in endothelial
cells, fluid flow alters activation of NF-
B resulting in changes in
expression of cell adhesion molecules (22-24). Therefore, we
hypothesized that fluid flow-induced shear stress alters
TNF-
-induced NF-
B activation and expression of cell adhesion
molecules in osteoblastic cells. To explore our hypothesis osteoblastic
UMR106 cells were exposed to OFF in the presence or absence of TNF-
. Our results suggest that OFF inhibits TNF-
-induced activation of
NF-
B.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All reagents were obtained from Sigma
except as otherwise noted.
Fluid Flow Experiments--
UMR106 cells were grown on glass
slides in minimal essential medium (Life Technologies, Inc., Rockville,
MD) supplemented with 10% fetal bovine serum (HyClone, Logan, UT).
Nearly confluent cells were incubated in minimal essential medium
without fetal bovine serum for 24 h before experiments. OFF was
generated as previously described with some modification (6). In brief, cells on glass slides were mounted in a parallel plate flow chamber attached to a custom designed fluid pump via rigid wall tubing. Cells
were exposed to OFF, in the absence or presence of 1 ng/ml TNF-
, for
various lengths of time at flow rates of 0, ±4, ±10, and ±20 ml/min
at a frequency of 1 Hz. In our flow chamber, these flow rates induce
peak shear stresses of 0, 1.9, 4.7, and 9.3 dyne/cm2,
respectively. Cells were then harvested for nuclear and cytosolic protein extraction or total RNA extraction.
Cell Viability and Adhesion Assay--
Cells were subjected to
OFF in the absence or presence of 1 ng/ml TNF-
at various flow rates
for 2 h. After exposure to OFF, cells adhering to glass slides
were washed twice with 10 ml of phosphate-buffered saline (PBS) without
Ca2+ and Mg2+ (PBS (
); Life Technologies,
Inc.) and were collected by trypsinization. Cell viability was
evaluated by a trypan blue dye exclusion test.
Preparations of Nuclear and Cytosolic Protein
Extracts--
Nuclear and cytosolic protein extracts were prepared as
described previously with some modification (17). After being washed twice with 10 ml of PBS (
), the cells were incubated on ice for 5 min
in 1 ml of PBS (
) containing 2 mM EDTA (pH 8.0),
harvested by scraping with a cell scraper, and then pelleted by
centrifugation at 12,000 × g for 15 s at 4 °C.
The cell pellet was resuspended in 100 µl of Buffer A (25 µg/ml
aprotinin, 1 mM dithiothreitol (DTT), 10 mM
HEPES-KOH (pH 7.9), 10 mM KCl, 10 µg of leupeptin, 1.5 mM MgCl2, 100 nM pepstatin, 1 mM phenylmethylsuflonyl fluoride, 50 mM NaF,
0.5 mM Na3VO4, 1 mM
sodium pyrophosphate, 5 µg/ml
N-tosyl-L-phenylalanine chromethyl ketone, and
0.4% Nonidet P-40 (Fluka, Milwaukee, WI)) and lysed by incubating on
ice for 10 min and then centrifuged at 12,000 × g for
5 min. The supernatant was used as cytosolic protein extract. The
pellet was washed with PBS (
), resuspended in 100 µl of Buffer C (1 mM DTT, 2 mM EDTA (pH 8.0), 20% glycerol, 20 mM HEPES-KOH (pH 7.9), 0.4 M KCl, 100 nM pepstatin, and 1 mM phenylmethylsuflonyl
fluoride), and lysed by freezing and thawing. After centrifugation at
12,000 × g for 5 min at 4 °C, the supernatant was
used as nuclear protein extract. Protein concentration was determined
by a microassay kit (Bio-Rad, Hercules, CA) using bovine serum albumin
as a standard. The nuclear and cytosolic protein extracts were
aliquoted and stored at
80 °C until analysis.
Electrophoretic Mobility Shift Assay--
Nuclear extracts (20 µg of protein) were used for electrophoretic mobility shift assay.
The NF-
B consensus oligonucleotide was obtained from Promega
(Madison, WI). The NF-
B oligonucleotide was labeled by T4
polynucleotide kinase in the presence of 20 µCi of
[
-32P]ATP (Amersham Pharmacia Biotech) and used
as a probe. To identify the NF-
B subunits, supershift analysis was
performed using antibodies directed against p50 and p65 (Santa Cruz
Biotechnology, Santa Cruz, CA). The antibodies were added to the
binding reaction mixture (40 mM HEPES-KOH (pH 7.9), 75 mM KCl, 0.5 M EDTA (pH 8.0), 0.5 mM
DTT, and 10% glycerol) before the addition of the labeled probe and
incubated for 1 h at 4 °C. Samples were loaded on 4%
polyacrylamide gels (29:1, acrylamide:bisacrylamide) containing 45 mM Tris-HCl (pH 8.0), 45 mM boric acid, and 1 mM EDTA (pH 8.0) for 3 h at 180 V. The gels were dried
and autoradiographed to Kodak X-Omat AR films at room temperature.
Northern Blot Analysis--
Total RNA was extracted using an
RNeasy kit (Qiagen, Valencia, CA). 15 µg of total RNA, as determined
by a spectrophotometer, was fractionated in 1% agarose-formaldehyde
gels. The RNA was transferred onto a nylon membrane (Gene Screen Plus;
PerkinElmer Life Sciences) by capillary action. The membrane was
prehybridized in a solution containing 30% deionized formamide, 50 mM sodium phosphate (pH 7.4), 1% SDS, and 1% bovine serum
albumin for 10 min at 55 °C. The heat-denatured probe for rat ICAM-1
cDNA was labeled with 50 µCi of [
-32P]dCTP
(Amersham Pharmacia Biotech) using a random primed DNA labeling kit
(Roche Molecular Biochemicals). The labeled cDNA probe was added to
the solution, and the hybridization was performed for 20 h at
55 °C. After the hybridization, the membranes were washed with 50 mM sodium phosphate (pH 7.4) and 1% SDS twice for 5 min at
55 °C. Then the membranes were autoradiographed to films at
80 °C. The mRNA levels were normalized to GAPDH mRNA
levels. Radioactivity of the band for the respective mRNA was
quantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis--
The cytosolic extracts containing 60 µg of protein were mixed with 1 volume of SDS loading buffer
containing 5% mercaptoethanol. After denaturing in boiling water for
10 min, the samples and molecular weight markers (Low or High; Bio-Rad)
were fractionated on 10 or 6% SDS polyacrylamide gels and
electroblotted onto membranes (Trans-Blot; Bio-Rad) using the
Mini-Protean II system (Bio-Rad). The membranes were soaked for 30 min
in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl,
and 0.05% Tween 20) containing 3% skim milk. Then the membranes were
incubated for 3 h with anti-I
B
, I
B kinase (IKK)
, or
IKK
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted
1:500 with TBST containing 3% skim milk. After washing three times
with TBST, the membranes were incubated for 1 h with anti-rabbit
IgG linked to horseradish peroxidase (Jackson ImmunoResearch,
West Grove, PA) diluted 1:3000 with TBST containing 3% skim milk.
After three additional washes with TBST, the membranes were soaked in
enhanced chemiluminescence detection reagents (ECL; Amersham Pharmacia
Biotech) according to the manufacturer's protocol. The membranes were
then exposed to films.
IKK in Vitro Kinase Assay--
The cytosolic extracts containing
200 µg of protein were preincubated with 1 µg of IKK
antibody
(Santa Cruz Biotechnology) for 1 h and were then incubated for
20 h, together with 20 µl of protein A/G-agarose (Santa Cruz
Biotechnology) at 4 °C. After washing 4 times with PBS (
),
one-half of each of the immunocomplexes were subjected to IKK kinase
assays. Kinase assays were performed as described previously with some
modification (25). Briefly, each immunoprecipitate was resuspended in
25 µl of kinase buffer (10 µM ATP, 2 µCi of
[
-32P]ATP, 25 µg/ml aprotinin, 1 mM
benzamidine, 1 mM DTT, 10 mM
-glycerophosphate, 20 mM HEPES-KOH (pH 7.9), 10 µg/ml
leupeptin, 2 mM MgCl2, 2 mM MnCl2, 10 mM p-nitrophenyl
phosphate, 2 µg/ml pepstatin, 0.5 mM phenylmethylsuflonyl
fluoride, 10 mM NaF, and 0.5 mM
Na3VO4) in the presence of 2.5 µg of I
B
(1-317; Santa Cruz Biotechnology) as a substrate at 30 °C for 30 min. Reactions were stopped by the addition of 6× SDS loading buffer
containing 30% mercaptoethanol. Samples and low molecular weight
markers were fractionated on 10% SDS polyacrylamide gels. The gels
were dried and autoradiographed to film at room temperature.
Radioactivity of the band for the respective IKK activity was
quantified by PhosphorImager. To confirm the presence of IKK, the
remaining one-half of each of the immunocomplexes was subjected to
Western blot analysis using 1:500 dilutions of IKK
or IKK
antibody.
Statistical Analyses--
Results are expressed as mean ± S.E. Statistical analysis was performed by analysis of variance with a
Bonferroni test. A p value less than 0.05 was considered significant.
 |
RESULTS |
OFF Does Not Alter the Viability of UMR106 Cells Adhering to Glass
Slides--
Trypan blue dye exclusion tests indicated that increasing
flow rates up to ± 20 ml/min, inducing shear stresses up to 9.3 dyne/cm2, tended to decrease the number of viable cells
adhering to glass slides (Fig. 1).
However, over 90% of cells were viable after exposure to OFF, which
was not significantly different from untreated controls. Moreover,
TNF-
did not affect cell viability relative to cells untreated with
TNF-
at any flow rates examined. Thus, under our study conditions
TNF-
was not cytotoxic.

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Fig. 1.
Cell viability and adhesion assay.
UMR106 cells were exposed to OFF at various flow rates for 2 h in
the absence or presence of 1 ng/ml TNF- . After exposure to OFF, cell
viability was evaluated by a trypan blue dye exclusion test. Data are
expressed as percentage of viable cells relative to untreated control.
Values are expressed as mean ± S.E. (n = 5). No
statistically significant differences were observed.
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OFF Inhibits TNF-
-induced Activation of NF-
B--
We first
examined the effect of OFF on NF-
B-DNA binding activity, in the
absence or presence of TNF-
, by electrophoretic mobility shift assay
using nuclear protein extracts obtained from UMR106 cells. A single
weak NF-
B-DNA complex was observed even in untreated control cells
(Fig. 2A, lane 1).
Exposure to OFF in the absence of TNF-
did not change this basal
activation of the NF-
B-DNA complex (lanes 2-4). In
contrast, activation of two distinct complexes was observed in TNF-
treated cells, namely a fast-migrating complex, which exhibited the
same mobility and weak activity as the complex observed in untreated
cells, and an additional slow-migrating complex, which exhibited
stronger activity (lane 5). Exposure to OFF decreased
dramatically the TNF-
-induced activation of the slow-migrating
NF-
B-DNA complex in a shear stress-dependent manner
(lanes 6-8). However, the activity of the fast-migrating
NF-
B-DNA complex was not altered by TNF-
or OFF. Time course
studies (Fig. 2B) revealed that treatment with TNF-
resulted in a rapid (within 15 min) increase of the slow-migrating
NF-
B-DNA complex, which continued throughout the 120-min treatment
period. Once again, TNF-
did not affect the activation of the
fast-migrating NF-
B-DNA complex. In contrast, exposure to OFF
decreased markedly the TNF-
-induced activation of the slow-migrating
NF-
B-DNA complex in a time-dependent manner, whereas it
did not change the activation of the fast-migrating NF-
B-DNA
complex.

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Fig. 2.
Effects of OFF on
NF- B activation. The open and
closed arrowheads indicate the fast- and slow-migrating
NF- B-DNA complexes, respectively. A, the effect of OFF
for 60 min on the activation of NF- B in the absence or presence of 1 ng/ml TNF- at various shear stresses as indicated in the figure. *
indicates excess labeled probes that did not bind to NF- B. These
results are typical of two other experiments. B, the results
of time course studies of NF- B activation. A similar result was
obtained from a separate experiment. C, the characterization
of NF- B subunits in UMR106 cells in the absence or presence of 1 ng/ml TNF- for 60 min. Supershift analyses performed by employing
antibodies directed against p50 and p65. # indicates the supershift
complex.
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|
We next characterized the NF-
B-DNA binding complexes in UMR106 cells
utilizing supershift analysis with specific antibodies against p50 and
p65, two of the more common members in the NF-
B/Rel family that form
a dimer in rat osteoblastic cells (17). A weak single NF-
B-DNA
complex in untreated cells (Fig. 2C, lane 1) was
supershifted by anti-p50 antibody (lane 2) but not by
anti-p65 antibody (lane 3), indicating that the complex
represents a p50 homodimer NF-
B. On the other hand, anti-p50
antibody supershifted both fast- and slow-migrating complexes induced
by TNF-
(lane 5), whereas anti-p65 antibody supershifted
only the slow-migrating complex (lane 6). Thus, the slow-
and fast-migrating NF-
B-DNA complexes represent a p50-p65
heterodimer and a p50 homodimer NF-
B, respectively.
OFF Inhibits a TNF-
-induced Increase in ICAM-1 mRNA
Expression--
ICAM-1 gene expression is mainly regulated by NF-
B
in rat osteoblastic cells (17, 18). We therefore examined ICAM-1
mRNA expression to evaluate the effect of OFF on the expression of an endogenous NF-
B target gene. Basal ICAM-1 mRNA expression was
detected even in the absence of TNF-
(Fig.
3A). This basal mRNA level
was not altered by a 2-h exposure to OFF at any shear stress level
examined. In contrast, treatment with TNF-
markedly increased ICAM-1
mRNA expression within 2 h. Exposure to OFF inhibited this
TNF-
-induced ICAM-1 mRNA expression in a shear
stress-dependent manner with the inhibition reaching
statistical significance at 4.3 dynes/cm2. Time course
studies (Fig. 3B) revealed that TNF-
dramatically induced
ICAM-1 mRNA expression during a 4-h exposure (lanes 1-5) while increasing the length of OFF exposure time decreased
TNF-
-induced ICAM-1 mRNA levels (lanes 4-9). GAPDH
mRNA levels were not altered by OFF regardless of the presence of
TNF-
.

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Fig. 3.
Effects of OFF on ICAM-1 mRNA
expression. The membranes were rehybridized with GAPDH cDNA
probe. Representative autoradiographs are shown. After the
radioactivity of the bands was measured by PhosphorImager, the ICAM-1
mRNA levels were normalized to GAPDH mRNA levels and then
expressed as a percentage of untreated control level. A, the
effect of OFF for 2 h on ICAM-1 mRNA expressions in the
absence or presence of 1 ng/ml TNF- at various shear stress levels
as indicated in the figure. Values are expressed as mean ± S.E.
(n = 3). *, statistically significant versus
control; #, statistically significant versus TNF- alone.
B, the results of time course studies of ICAM-1 mRNA
expression. Values are expressed as mean ± S.E.
(n = 2).
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OFF Decreases TNF-
-induced I
B
Degradation--
To
evaluate the mechanism by which OFF affects NF-
B activation, we
examined protein levels of I
B
, which is the only endogenous inhibitor of NF-
B activation thus far identified. I
B
protein was highly expressed in the cytosol in the absence of TNF-
(Fig. 4, upper panel, lane
1). Treatment with TNF-
markedly decreased I
B
protein
levels within 60 min (lane 2), reflecting a degradation of
I
B
. Exposure to OFF in the presence of TNF-
resulted in a
substantial increase in I
B
protein levels in a shear
stress-dependent manner (lanes 3-5). I
B
protein levels in cells exposed to TNF-
and OFF at 9.3 dynes/cm2 were similar to untreated control levels. These
results suggest that OFF-induced shear stress decreases TNF-
-induced
I
B
degradation. Protein levels of IKK
and IKK
, two kinase
members of the IKK family that regulate I
B
phosphorylation and
thus degradation (21, 26-28), were not affected by exposure to TNF-
or OFF (Fig. 4, middle and lower panels,
respectively).

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Fig. 4.
Effects of OFF on
I B ,
IKK , and IKK protein
levels. 60 µg of cytosolic protein extracts were subjected to
Western blot analysis. Cell treatments are indicated in the figure.
Positions of molecular mass markers, carbonic anhydrase (35 kDa), ovalbumin (50 kDa), and bovine serum albumin (87 kDa), are
indicated. These results are typical of two other experiments.
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Effect of OFF on IKK Activation--
To evaluate the mechanism by
which OFF decreases TNF-
-induced I
B
degradation and NF-
B
activation, we examined the activities of endogenous IKK utilizing
I
B
as a substrate in an in vitro kinase assay. The
weak IKK activation observed in untreated cells (Fig.
5A, lane 1)
dramatically increased after a 30-min exposure to TNF-
(lane
2). Exposure to OFF at 9.3 dyne/cm2 significantly
inhibited this TNF-
-induced IKK activation (lane 3). OFF
decreased the TNF-
-induced IKK activation in a shear stress-dependent manner (Fig. 5B). Time course
studies revealed that TNF-
markedly increased IKK activation
throughout the 60-min treatment period (Fig. 5C, lanes
1-4). Additionally exposure to OFF decreased TNF-
-induced IKK
activation to a greater degree with increasing exposure duration
eventually reaching control levels by 60 min (lane 5-7).
Middle and lower panels in Fig. 5 represent the
protein levels of IKK
and IKK
analyzed by Western blot analysis
using a cytosolic immunocomplex precipitated by anti-IKK
antibody to
confirm the presence of IKK. The immunoprecipitated protein levels of
both IKK
and IKK
did not change following exposure to TNF-
or
OFF.

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Fig. 5.
Effects of OFF on IKK activation. 200 mg
of cytosolic protein extracts were immunoprecipitated by anti-IKK
antibody, one-half of each immunocomplex was subjected to IKK in
vitro kinase assays with I B as a substrate, and the
remaining one-half of each immunocomplex was subjected to Western blot
analysis using anti-IKK and IKK antibodies. The radioactivities
for IKK activities were measured by PhosphorImager and were expressed
as a percentage of untreated control level. A, the effect of
OFF at 9.3 dyne/cm2 for 60 min on IKK activations in the
absence or presence of 1 ng/ml TNF- . Values are expressed as
mean ± S.E. (n = 3). *, statistically significant
versus control; #, statistically significant
versus TNF- alone. B, the effect of OFF on IKK
activity at various shear stress levels as indicated in the figure.
Values are expressed as mean ± S.E. (n = 2).
C, the results of time course studies of IKK activation.
Values are expressed as mean ± S.E. (n = 2).
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|
 |
DISCUSSION |
The present study demonstrates for the first time that fluid flow
has an inhibitory effect on NF-
B-DNA binding activity in the
nucleus, especially on the activation of the slow-migrating NF-
B-DNA
complex induced by TNF-
. The results of super shift analysis clearly
indicate that the slow-migrating NF-
B-DNA complex activated by
TNF-
is the p50-p65 heterodimer NF-
B and the basal NF-
B-DNA
complex activated constitutively in UMR106 cells is the p50 homodimer
NF-
B. TNF-
-induced ICAM-1 mRNA expression was also inhibited
by OFF as shown by Northern blot analysis. The inhibitory effects of
OFF on both TNF-
-induced NF-
B activation and ICAM-1 mRNA
expression were shear stress-dependent and also increased
with OFF exposure duration. These results suggest that OFF has a potent
biophysical effect on mechanotransduction pathways, especially at the
transcriptional level in osteoblastic cells stimulated by TNF-
.
Theoretical models predict that the wall fluid shear stresses in the
canaliculi of bone tissues are 6-30 dynes/cm2 (3). The
maximum flow rate used for our study was ± 20 ml/min, and
frequency was 1 Hz. This fluid flow induces peak shear stresses of 9.3 dyne/cm2 in our flow chamber. Thus, the fluid shear stress
generated by our OFF system is within the range bone cells experience
in vivo. Under the condition of our studies, fluid flow had
no effect on cell viability or adhesion.
It has been demonstrated that the p50-p65 heterodimer NF-
B is
capable of transactivating gene expression (29). Indeed, we previously
have shown that TNF-
induces interleukin-6 and ICAM-1 gene
expressions in rat osteoblastic cells via activation of the p50-p65
heterodimer NF-
B (17, 18). The shear stress and time course for OFF
inhibition of TNF-
-induced ICAM-1 mRNA expression were similar
to the shear stress and time course for the inhibition of
TNF-
-induced NF-
B activation. Taken together with our previous
findings, this suggests the possibility that the effect of OFF on
TNF-
-induced ICAM-1 gene expression may be via an
NF-
B-dependent pathway. However, a direct link between OFF, TNF-
, and ICAM-1 expression cannot be made without rigorous mutational analysis. In any case, our data clearly show an interaction of OFF with TNF-
that has a strong potential to alter bone cell activity.
In this study, neither OFF nor TNF-
affected the basal p50 homodimer
NF-
B activation. Moreover, basal ICAM-1 mRNA expression, which
was detected in the absence of TNF-
, was not altered by OFF. It has
been reported that moderate levels of p50 homodimer NF-
B activation
was conserved in other cells (30-32). The role for the p50 homodimer
NF-
B activation in constitutive-type transcription is unclear, but
it may provide low levels of transcriptional activity or it may serve
as a transcriptional repressor protein (19, 33, 34).
In response to external stresses, mammalian cells rapidly translocate
NF-
B to the nucleus. Once there, this protein binds to 10-base pair
B sites as a dimer within the DNA of specific genes, resulting in
the regulation of transcription of these genes. The activity of NF-
B
is tightly regulated by interactions with inhibitory I
B proteins in
the cytoplasm, which block transport of NF-
B into the nucleus in the
absence of activating signals. Most extracellular signals such as
TNF-
activate NF-
B through a common pathway dependent on
phosphorylation-induced degradation of I
B (for reviews, see Refs.
19, 20, and 35). In this study, we demonstrated that cytosolic I
B
protein levels were dramatically decreased by TNF-
and that OFF
attenuated the inhibitory effect of TNF-
on I
B
.
Recent evidence suggests that I
B
degradation is regulated by
phosphorylation via IKK (36). IKK is a protein complex the catalysis of
which is generally carried out by a heterodimeric kinase consisting of
IKK
and IKK
subunits (21, 26-28). Indeed, we observed IKK
, as
well as IKK
, by Western blot analysis using the immunocomplex
precipitated by anti-IKK
antibody. This suggests that the IKK
complex within the cytosol of UMR106 cells is largely a heterodimeric
complex of IKK
and IKK
. Both IKK
and IKK
activities are
stimulated in response to TNF-
or interleukin-1 (21, 26-28, 37).
Furthermore, knockout mice studies indicate that IKK
plays an
important role in skeletal development (38). Therefore, we examined IKK
kinase activity to evaluate the mechanism by which OFF affects
TNF-
-induced I
B
degradation and NF-
B activation. Exposure
to OFF inhibited TNF-
-induced activation of IKK in a shear
stress-dependent manner. The shear
stress-dependent pattern of OFF inhibition of
TNF-
-induced IKK activation was similar to the shear
stress-dependent pattern of the inhibition of
TNF-
-induced NF-
B activation, ICAM-1 mRNA expression, and
I
B
degradation, suggesting a link between these signaling
pathways and OFF. Neither TNF-
nor OFF affected IKK
and IKK
levels, suggesting that the effects we observed were not because of an
effect on protein synthesis or degradation. Our results suggest that
IKK is an initial target molecule for OFF effects on osteoblastic
cells. OFF inhibits TNF-
-induced IKK activation, leading to a
decrease in phosphorylation and degradation of inhibitory I
B
,
which in turn results in the decrease of TNF-
-induced NF-
B
activation and the transcription of target genes.
The precise mechanism by which OFF decreases TNF-
-induced IKK
activity remains unclear. Recently it has been demonstrated that IKK
itself is also phosphorylated and regulated by one or more upstream
kinases (37). One possibility is that OFF affects dephosphorylation
events by inducing conformational changes in these upstream kinases of
IKK. Shear stress might lead directly to dephosphorylation by deforming
kinases and inactivating them (39). On the other hand, it has been
demonstrated that fluid flow rapidly activates mitogen-activated
protein kinases, including extracellular signal-regulated kinase and
c-Jun N-terminal kinase, both recognized stress-activated protein
kinases (40-44), and focal adhesion kinase (45) in vascular
endothelial cells. Therefore, another possibility is that OFF might
activate upstream kinases, leading to phosphorylation of IKK itself and
resulting in decreased IKK activation. Indeed, more recently it was
discovered that phosphorylation of IKK
at C-terminal serines can
also result in negative regulation of IKK activity by changing the
conformation of the intrinsic kinase activator domain (46, 47). Another
possibility is an indirect effect through which OFF-induced shear
stress may modify distinct signaling factors that compete for or couple
with TNF-
-induced IKK activation pathways.
An important point to consider is that our results were obtained with
UMR106 cells, a rat osteogenic osteosarcoma cell line with osteoblastic
characteristics. Although this cell line has provided many important
insights into bone cell biology, especially as related to
mechanotransduction, it could be argued that transcription factors in
UMR106 cells are not the same as those in authentic osteoblasts.
However, we believe that this is unlikely, because it has recently been
shown that NF-
B is required for TNF-
activity in primary culture
human osteoblasts (48).
In summary, our results suggest that OFF inhibits TNF-
-induced
NF-
B activation in an osteoblastic cell line. Previous studies suggest that TNF-
increases accumulation of bone resorption
stimulating cytokines by osteoblastic cells (12, 13) and may also
stimulate osteoblastic differentiation, both of which may be mediated
by NF-
B. (49) Thus, OFF may modulate bone turnover through its effect on bone cell activity.