From the Division of Bone and Mineral Diseases and § Department of Pathology, Washington University School of Medicine and Barnes-Jewish Hospital, St. Louis, Missouri 63110
Received for publication, November 29, 2000
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ABSTRACT |
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The differentiation of cells of the monocytic
lineage into mature osteoclasts (OC) is specifically induced by the
tumor necrosis factor-related factor, RANKL
(receptor activator of NF- The differentiation of osteoclast
(OC)1 precursors of the
monocytic lineage into mature OCs is induced specifically by
simultaneous stimulation of bone marrow monocytes by two cytokines
produced by bone marrow stromal cells, M-CSF (1-3) and a TNF-related
factor known as receptor activator of NF- It is now recognized that inhibition of osteoclastogenesis is the main
mechanism by which estrogen (E2) prevents bone loss (9, 10). Moreover,
considerable evidence support the hypothesis that E2 down-regulates OC
formation by blunting the production of IL-1, IL-6, and TNF (9-13),
cytokines that enhance the stromal cell production of RANKL and
M-CSF (14-17). Additional mechanisms that serve to explain the
antiosteoclastogenic effects of E2 include the ability of sex steroids
to stimulate the production of osteoprotegerin (OPG), a decoy
RANKL receptor (18), and a direct repressive effect on the production
of membrane-bound M-CSF (19, 20).
Despite the relevance of RANKL as an osteoclastogenic factor, and of
the potent antiosteoclastogenic effect of E2, sex steroids have not
been reported to regulate the production of RANKL. The lack of such
regulation is consistent with the absence of both estrogen-responsive
elements and binding sequences for E2-regulated transcription factors
on the RANKL promoter (21).
Binding of RANKL to its signaling receptor RANK results in the
activation of Jun N-terminal kinase (JNK), a mitogen-activated protein kinase that increases the trans
activation activity and the production of AP-1 transcription factors.
We have recently reported that E2 down-regulates TNF-induced JNK
activation in monocytic cells (22), and others have shown that OC
formation is impaired in mice lacking the AP-1 factors c-Fos and c-Jun
(23, 24). These data prompted us to investigate the hypothesis that an
additional mechanism by which E2 down-regulates OC formation is by
decreasing the responsiveness of maturing OC to RANKL.
Here we show that E2 decreases the activation of JNK leading to
decreased production of the osteoclastogenic transcription factors
c-Fos and c-Jun. Thus, E2 down-regulates OC formation not only by
modulating the production of osteoclastogenic cytokines but also by
affecting the sensitivity of maturing OC to RANKL.
All animal procedures were approved by the Animal Care and Use
Committee of Barnes-Jewish Hospital. Unless otherwise specified, reagents and media were purchased from Sigma.
RAW 264.7 Cell and BMM Cultures--
Bone marrow monocytes
(BMM), defined as CD11b+ cells, were purified from the bone
marrow of 5-week-old mice by positive immunoselection using MACS CD11b
immunomagnetic beads (Milteny Biotec, Auburn, CA). RAW 264.7 cells were
obtained from the ATCC. All cells were maintained in phenol red-free
media and 10% charcoal-stripped serum cultured in
In other experiments the MEK1/ERK pathway inhibitor PD 98059 (New
England Biolabs, Beverly, MA) was added (50 µM) to
control and E2-treated RAW 264.7 cells 1 h before stimulation with
RANKL for 30 min.
OC Generation and Characterization--
OC were generated by
culturing BMM with recombinant soluble RANKL (20 ng/ml) and M-CSF (10 ng/ml) for 7 days as described (4, 6). OC were also generated by
culturing RAW 264.7 cells with RANKL (20 ng/ml) without M-CSF as
described (25). Both culture systems generate large numbers of
TRAP+ multinucleated cells, which express typical OC
markers including calcitonin and vitronectin receptors and positivity
for pp60c-src and cathepsin K (4, 6, 25).
At the end of each experiment, cells were then fixed and stained for
tartrate-resistant acid phosphatase (TRAP). Expression of calcitonin
receptors and the ability to form resorption pits in vitro
were assessed as previously described (26). Immunohistochemical staining for pp60c-src and cathepsin K was
conducted as described (27). More than 98% of the TRAP+
cells with three or more nuclei showed specific binding of labeled calcitonin and expression of pp60c-src and
cathepsin K. These cells were capable of forming resorption pits
in vitro. Therefore, TRAP+ cells with three or
more nuclei were defined and counted as OC.
JNK Activity Assay and Western Blot Analysis of Total and Active
JNK--
RAW 264.7 cells were treated with E2
(10
The levels of dephosphorylated and phosphorylated species of JNK1 and
JNK2 (total JNK1/2) and those of the active (phosphorylated) forms of
JNK1 and JNK2 (active JNK1/2) were measured using whole cell lysates
from control and E2-treated RAW 264.7 cells either unstimulated or
stimulated with RANKL for 30 min. Cell lysates (50 µg) were resolved
on 12% SDS-PAGE and transblotted onto nitrocellulose. Total and
phosphorylated JNK1/2 were detected using antibodies specific for
either total JNK1 and JNK2 or phosphorylated JNK1 and JNK2 (Santa Cruz
Biotechnology) using an ECL detection system (Amersham Pharmacia Biotech).
Western Blot Analysis of Total and Active MKK4--
Western blot
studies were conducted using whole cell lysates from control and
E2-treated RAW 264.7 cells either unstimulated or stimulated with RANKL
for 30 min. Cells were washed twice with ice-cold phosphate-buffered
saline and then lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH.8, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40 (v/v), 1 mM NaF, 10 µg/ml leupeptin, 2 mM Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride) as described (28). Proteins were boiled
in Laemmli loading dye, resolved on 12% SDS-PAGE, and transblotted
onto nitrocellulose. The levels of total and phosphorylated MKK4 were
detected using antibodies (New England Biolabs) specific for
phosphorylated (active) and total (dephosphorylated plus
phosphorylated) MKK4 using an ECL detection system (Amersham Pharmacia Biotech).
Extraction of Nuclear Protein and Electrophoretic Mobility Shift
Assays--
Electrophoretic mobility shift assays were performed by
incubating nuclear extracts prepared from control and E2-treated RAW 264.7 cells stimulated with RANKL (20 ng/ml) for 1 h with a
32P-labeled double-stranded probe (5'
AAAGCAGCAGCCTGAGCTCATGATCA 3') synthesized to represent the
AP-1 binding sequence (underlined) as described previously (22).
Monoclonal antibodies directed against members of the Jun and Fos
families of nuclear proteins were used to supershift the relevant proteins.
The probe was end-labeled with T4 polynucleotide kinase according to
manufacturer's instructions (Promega, Madison, WI). The annealed probe
was incubated with nuclear extract on ice for 30 min prior to
separation of the DNA-protein complexes on 4% nondenaturing PAGE, pre-run for 1 h in 0.25× TBE running buffer. The
gels were dried and exposed to Kodak XAR-5 film for the appropriate
length of time. For band supershifting, 200 ng of the appropriate
antibody was added to the reaction mixture on ice for 40 min before
adding the oligonucleotide probe.
Western Blot Analysis of Jun and Fos--
Western blot analysis
was performed using antibodies specific for either c-Jun, JunD, JunB,
c-Fos, FosB, Fra-1, or Fra-2 (Santa Cruz Biotechnology) as described
(22).
Statistical Analysis--
Group mean values were compared by
two-tailed Student's t test or one-way analysis of variance
as appropriate. Subsequent mean comparison tests were performed by the
Fisher protected least significant difference test.
E2 Decreases RANKL-induced Osteoclastogenesis--
The discovery
that stimulation with RANKL and M-CSF is sufficient to induce the
differentiation of BMM into OC in the absence of stromal cells (4, 5)
has made it possible to investigate whether E2 has direct effects on
maturing hematopoietic OC precursors. Triplicate experiments revealed
that E2 (10
Inhibition of either M-CSF- or RANKL-induced signals could account for
the ability of E2 to blunt the differentiation of BMM into OC. Because
we wished to examine the role of E2 in modulating RANKL-induced
signals, we utilized the property of RAW 264.7 cells, a murine
monocytic line, to differentiate into OC when treated with RANKL in the
absence of M-CSF (25). Quadruple experiments (Fig. 1B)
revealed that, similar to its effect on primary BMM, E2 decreases by
~40% the differentiation of RAW 264.7 cells into OC induced by
RANKL. The selective estrogen receptor modulators 4-hydroxytamoxifen
and raloxifene were as effective as estrogen in suppressing
RANKL-induced differentiation of both BMM and Raw 264.7 cells into OC.
Moreover, the inhibitory effects of estrogen were reversed by the
estrogen antagonist ICI 182780 (3 1), a finding that suggests
sex steroids have a genomic effect on OC precursors.
Thus, the inhibitory effect of E2 on RANKL-induced signals is
sufficient to block OC formation, and RAW 264.7 cells represent an
appropriate model for dissecting the molecular mechanisms involved.
E2 Down-regulates MKK4-induced JNK Activation--
A key signaling
event induced by the binding of RANKL to RANK is the activation of JNK
(25). To investigate whether E2 decreases JNK activity, RAW 264.7 cells
were treated with either E2 or control vehicle for 24 h, a length
of time sufficient to induce genomic effects, and then stimulated with
RANKL for 0-30 min. Nuclear extracts were then immunoprecipitated
using an antibody that recognizes the two members of the JNK family,
JNK1 and JNK2. Equal amounts of proteins were recovered and incubated
with recombinant GST-c-Jun (a JNK substrate) and
[
Western blot analysis of RAW 264.7 cell extracts with an antibody that
recognizes the phosphorylated (active) forms of JNK1 and JNK2 showed
that unstimulated cells have low levels of active JNK1 and undetectable
levels of active JNK2. RANKL induced the production of active JNK1 but
not of JNK2 (Fig. 2C). However, at all time points
phosphorylated JNK1 was ~50% lower in E2-treated cells than in cells
treated with vehicle. E2 also had a small inhibitory effect on the
levels of phosphorylated JNK1 in unstimulated cells. In contrast,
Western blot analysis by an antibody recognizing both the
dephosphorylated and the phosphorylated species of JNK1 and JNK2 (total
JNK1/2) revealed the presence of both JNK species in unstimulated
cells. However, neither RANKL nor E2 altered the level of total JNK1
and JNK2 (Fig. 2D). Together, these findings demonstrate
that E2 represses the ability of RANKL to induce the activation of JNK1.
MKK4 is a kinase known to activate JNK1 by inducing its phosphorylation
(32). Analysis of the effects of E2 on MKK4 using an antibody specific
for the phosphorylated (active) form of this kinase revealed (Fig.
3A) that RANKL increases by
~3-fold the level of phosphorylated MKK4 and that E2 decreases by
~50% the activation of MKK4 induced by RANKL. E2 also had a small
inhibitory effect on the levels of phosphorylated MKK4 in unstimulated
cells. In contrast, Western blot studies conducted using an antibody that recognizes both the dephosphorylated (inactive) and the
phosphorylated species of MKK4 revealed that neither RANKL nor E2
modulates the total levels of MKK4 (Fig. 3B). Thus, although
the signaling pathway linking RANKL to MKK4 remains to be determined,
the data demonstrate that E2 blunts MKK4 and JNK activation, thus
leading to diminished JNK activity.
MKK4 is activated by MEKK1, a kinase that also leads to the
phosphorylation of ELK-1 through the MEK1/ERK pathway (33, 34). Thus, a regulatory effect of E2 on MEKK1 could lead to blunted ERK
activity and decreased ERK-induced production of c-Fos. The hypothesis
that E2 blunts nuclear levels of c-Fos via inhibition of the MEK1/ERK
pathway was excluded by demonstrating that PD 98059, a compound that
blocks MEK1 activation (35, 36), does not abolish the ability of E2 to
decrease nuclear levels of c-Fos (Fig.
4).
E2 Decreases the Nuclear Levels of c-Jun and c-Fos and the Binding
of These Proteins to DNA--
Because RANKL-induced JNK activation
results in enhanced production of Jun and Fos, we examined the effects
of E2 on the nuclear levels of these proteins. Western blot studies
conducted using RAW 264.7 nuclear extracts and monoclonal antibodies
specific for individual member of the Jun and Fos families revealed
that RANKL increased c-Jun levels and induced c-Fos production, peaking at 30 min. (Fig. 5). Moreover, in
RANKL-stimulated cells, the levels of both c-Jun and c-Fos were
~3-fold lower in those treated with E2 than in those treated with
control vehicle. Neither RANKL nor E2 affected the levels of JunB,
JunD, FosB, Fra-1, and Fra-2 (not shown).
Jun and Fos exert their transcriptional effects by binding to the AP-1
consensus sequence(s) in the promoter region of target genes. The
binding of these nuclear protein to DNA is proportional to their
nuclear concentration and phosphorylation status. To determine the
effects of E2 on the ability of Jun and Fos proteins to bind to DNA,
nuclear extracts from control and E2-treated RAW 264.7 cells were
incubated with a labeled oligonucleotide corresponding to the AP-1
consensus sequence. Samples from all cells generated a single
DNA-protein complex, which was confirmed as AP-1 by the ability of
50-fold molar excess AP-1 consensus oligonucleotide to displace
the complex. RANKL increased by ~3-fold the binding of AP-1 to its
binding motif (Fig. 6A).
Moreover, in samples from RANKL-treated cells, a specific anti-c-Jun
antibody led to the almost complete disappearance of the AP-1 band,
whereas anti-JunD and anti-JunB antibodies had no effects. The AP-1
complex was also supershifted by anti-c-Fos antibody (Fig.
5B), whereas antibodies against Fos-B, Fra-1, and Fra-2 had
no effect. Together, these findings demonstrate that RANKL increases
the binding to DNA of c-Jun/c-Fos and JunD/c-Fos heterodimers. These
experiments also revealed that E2 completely blocks the ability of
RANKL to increase the binding of c-Jun/c-Fos and JunD/c-Fos
heterodimers to DNA (Fig. 6, A and B).
It is now recognized that E2 prevents bone loss via multiple and
complex effects on bone marrow and bone cells, which results in
decreased OC formation, increased OC apoptosis, and decreased capacity
of mature OCs to resorb bone (9). E2 modulates OC apoptosis and OC
activity both directly (37, 38) and indirectly via regulation of growth
factors and prostaglandins (39, 40). Conversely, inhibition of OC
formation in vivo results, in part, from the ability of E2
to block the production of the pro-osteoclastogenic cytokines IL-1,
IL-6, and TNF (9-13). Another relevant effect of E2 in vivo
is its capacity to block the production of soluble M-CSF via an
IL-1- and TNF-dependent mechanism (16, 17, 41). In
vitro studies have also revealed that E2 directly block the differentiation of OC precursors into mature OC (42), although the
relevance of this phenomenon in vivo remains to be determined.
Although IL-1, IL-6, and TNF induce the formation of OCs in
vivo and in complex culture systems containing stromal cells, osteoblasts, and OC precursors, these cytokines (alone or in
combination) are not capable of inducing the formation of OCs when
added to purified OC precursors. Moreover, osteopetrosis is not a
feature of mice lacking the capacity to produce and/or respond to
either IL-1, IL-6, or TNF (11-13). These observations are consistent
with the notions that IL-1, IL-6, and TNF enhance OC formation by
increasing the production of M-CSF (43, 44) and RANKL or by
potentiating the osteoclastogenic effects of RANKL (45).
Binding of RANKL to RANK results in a cascade of intracellular events,
including the activation of the transcription factor NF- The relevance of the RANK/TRAF6/JNK/AP-1 signaling pathway is
demonstrated by the fact that osteopetrosis is a feature of TRAF6 (49)-
and c-fos-deficient mice (50, 51). Moreover, osteoclastogenesis is impaired in transgenic mice overexpressing a
c-Jun mutant that cannot be phosphorylated by JNK1 (24). In agreement
with these data and with recent observations of Shevde et
al. (42), we have found that E2 decreases the ability of RANKL to
activate MKK4 and JNK1 and the production of osteoclastogenic transcription factors c-Jun and c-Fos (23, 24). However, we recognize
that our findings do not conclusively demonstrate that MKK4 activation
is the mechanism by which RANKL and E2 regulate JNK activity and the
resulting production of c-Fos and c-Jun.
The mechanism by which E2 regulates the activation of MKK4 also remains
to be determined, although it is likely that E2 may regulate the
production of upstream signaling molecule(s) involved in the activation
of MKK4.
Because ligand binding to RANK activates NF- In summary, our findings demonstrate that E2 represses the ability of
RANKL to induce the activation of JNK1, thus leading to lower levels of
c-Fos and c-Jun and decreased bindings of these proteins to DNA. Thus,
a mechanism by which E2 blocks OC formation is by decreasing the
responsiveness of OC precursors to the osteoclastogenic cytokine
RANKL.
B
ligand; also known as OPGL, ODF, or TRANCE). Because inhibition of
osteoclastogenesis is one of the main mechanisms by which estrogen (E2)
prevents bone loss, it is likely that E2 may regulate either the
production of, or the target cell responsiveness to RANKL. We found
that E2 decreases the differentiation into OC of both murine bone
marrow monocytes and RAW 264.7 cells, a monocytic line, by
down-regulating the activation of Jun N-terminal kinase 1 (JNK1).
Diminished JNK1 activity results in decreased nuclear levels of the key
osteoclastogenic transcription factors, c-Fos and c-Jun, and lower
binding of these transcriptional inducers to DNA. Thus, one novel
mechanism by which E2 down-regulates osteoclastogenesis is by
decreasing the responsiveness of OC precursors to RANKL.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B ligand (RANKL) (also
known as OPGL, TRANCE, or ODF) (4-6). The formation of mature OCs is completely dependent on the presence of both RANKL and M-CSF, as
demonstrated by the lack of OC development in mice lacking the
expression of either factor (5, 7, 8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MEM and treated
with either E2 (10
8 M) or control
vehicle. For some experiments, RAW 264.7 cells were treated with
raloxifene (10
7 M),
4-hydroxytamoxifene (10
7 M), and
estrogen (10
8 M) plus ICI 182780
(10
6 M).
8 M), ICI 182780
(10
6 M), estrogen + ICI, 4-hydro-tamoxifen
(10
7 M) or raloxifene (10
7
M), or vehicle for 24 h and then stimulated with RANKL
(200 ng/ml) for 5-30 min. JNK activity was determined in cell extracts
as described (22). Briefly, cell extracts were precleared with 10-15
µl of protein A-Sepharose beads. The resin was then removed by
centrifugation and the supernatant incubated with 1 µg of anti-JNK antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 4 h on ice. The immunocomplexes were then precipitated using protein A-Sepharose beads. The beads were washed, resuspended in 25 µl of
kinase buffer containing 50 µM ATP and 5 µCi of
[
-32P]ATP, and incubated for 25 min at 30 °C with 1 µg of recombinant c-Jun (28, 29). The reactions were boiled in
Laemmli loading dye and resolved by 12% SDS-PAGE. Phosphorylated
c-Jun-GST protein was detected by autoradiography as described
previously (30). For some experiment, JNK activity was determined in
cell extracts using a JNK assay kit (Cell Signaling Technology,
Beverly, MA)as described by the manufacturer. Briefly, JNK was
precipitated by incubating cell extract overnight with c-Jun fusion
protein beads. Beads were resuspended in 50 µl of kinase buffer
supplemented with 100 µM ATP and incubated for 30 min at
30 °C. The reaction was boiled in SDS sample buffer, resolved on a
4-20% Tris-glycine gel, and electrotransferred to nitrocellulose
membrane. The appropriate primary and secondary antibodies were used
for detection of phosphorylated c-Jun on the membrane followed by a
standard chemiluminescence reaction using LumiGLO (Cell Signaling Technology).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8 M) decreases by
~40% the ability of soluble recombinant RANKL and M-CSF to induce
the differentiation of primary murine BMM into mature OC (Fig.
1A), defined as TRAP,
calcitonin receptor, pp60c-src, and
cathepsin K+ multinucleated cells capable of resorbing bone
in vitro.
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Fig. 1.
E2 decreases the number (mean ± S.E.) of OC generated by cultures of BMM (CD11b+ cells)
stimulated with both RANKL (20 ng/ml) and M-CSF (10 ng/ml) for
7 days (A) and RAW 264.7 cells
stimulated with RANKL only for 7 days (B). * = p < 0.05 compared with controls.
-32P]ATP. Phosphorylated material was resolved by
SDS-gel and visualized by autoradiography. These studies revealed (Fig.
2A) that RANKL increases JNK
activity 10-fold, peaking at 15 min. At all time points E2 treatment
blunted RANKL-induced JNK activity, as compared with cells treated with
RANKL plus E2 vehicle. Peak inhibitory activity (5-fold inhibition
compared with vehicle-treated controls) was found at 15 min. The
inhibitory effect of E2 on peak JNK activity was reversed by the
estrogen antagonist ICI 182780 (Fig. 2B). Raloxifene and
tamoxifen blocked RANKL-induced JNK activity in a manner similar to
E2.
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Fig. 2.
E2 decreases JNK activity and the ability of
RANKL to increase the level of active (phosphorylated) JNK1 in RAW
264.7 cells. A, time course experiments revealed that RANKL
increases JNK activity 10-fold, peaking at 15 min. B,
RANKL-induced JNK activity was repressed by raloxifene
(10 7 M), and 4-hydroxytamoxifen.
The inhibitory effects of estrogen were reversed by the estrogen
antagonist ICI 182780 (ICI) (10
6
M). C, E2 decreases the level of active
(phosphorylated) JNK1 in RANKL-stimulated RAW 264.7 cells. Active
(phosphorylated) JNK2 was not detectable in either unstimulated or
RANKL-stimulated RAW 264.7 cells. D, E2 does not
regulate the level of total (dephosphorylated + phosphorylated) JNK1
and JNK2. The figure shows representative data from one of three
replicate experiments.
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Fig. 3.
A, E2 blunts the ability of RANKL to
increase the level of active (phosphorylated) MKK4 in RAW 264.7 cells.
B, RANKL and E2 do not regulate the level of total
(dephosphorylated + phosphorylated) MKK4. The figure shows
representative data from one of three replicate experiments.
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Fig. 4.
Inhibition of the MEK1/ERK pathway by
PD 98059 does not abolish the ability of E2 to decrease
RANKL induced c-Fos production. 50 µM PD 98059 was
added to control and E2-treated RAW 264.7 cells 1 h before
stimulation with RANKL for 30 min. C-Fos levels were then measured in
nuclear extracts by Western blot conducted using a monoclonal antibody
specific for c-Fos. The figure shows representative data from one of
three replicate experiments. Densitometric analysis of the data from
all experiments demonstrated that PD 18057 does not abolish the
ability of E2 to decrease c-Fos levels.
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Fig. 5.
E2 decreases the nuclear levels of c-Jun and
c-Fos. Western blot analysis conducted using nuclear extracts from
untreated and E2-treated RAW 264.7 cells stimulated with RANKL for 30 min and monoclonal antibodies against specific Jun and Fos proteins.
Densitometric analysis of the data from all experiments demonstrated
that E2 decreases by ~3-folds c-Jun and c-Fos levels. E2 did not
regulate the levels of JunD, JunB, FosB, Fra-1, and Fra-2 (not shown).
The figure shows representative data from one of three replicate
experiments.
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Fig. 6.
E2 decreases the binding of c-Jun/c-Fos
heterodimers to an AP-1 consensus sequence. Electrophoretic
mobility shift assays were performed by incubating nuclear extracts
prepared from control and E2-treated RAW 264.7 cells stimulated with
RANKL for 1 h with a 32P-labeled double-stranded probe
synthesized to represent the AP-1 consensus sequence. Monoclonal
antibodies directed against members of the Jun (panel A) and
Fos families (panel B) of nuclear proteins were used to
supershift the relevant protein. The protein DNA complex was
supershifted by antibodies directed against c-Jun and c-Fos. Antibodies
against JunD, JunB, FosB, Fra-1, and Fra-2 had no effect. Densitometric
analysis of the data from three experiments revealed that AP-1 binding
to the probe was ~3-fold lower in E2-treated than in vehicle-treated
samples. The figure shows representative data from one of three
replicate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and the
stimulation of the protein kinase JNK (25, 46), which, in turn,
increases transcription of the c-jun and
c-fos genes (33, 47). This is because the transcription of
c-Jun is regulated by two AP-1 binding sites present in its promoter regions (33, 48). These sites are constitutively occupied by Jun/ATF2
heterodimers (33, 48), which generate a basal rate of gene
autostimulation. However, the magnitude of the autoinduction of the
c-jun gene by Jun/ATF2 heterodimers is enhanced by
JNK-dependent phosphorylation of two serine residues within
the N-terminal activation domain of c-Jun (33). JNK is also responsible
for the phosphorylation of ternary complex factors (such as Elk-1) that
are constitutively bound to the serum response element in the c-Fos
promoter (33). Because JNK-dependent phosphorylation of
ternary complex factors stimulates the transcription of the
c-fos gene, JNK activation results in increased
c-fos gene expression (33).
B and E2 represses
IL-6-induced NF-
B activation in stromal cells, it could be argued
that NF-
B may mediate, at least in part, the repressive effects of
E2 on OC development. The regulatory effects of E2 on RANKL-induced
NF-
B activation remain to be investigated. However, in agreement
with Hsu et al. (25), in preliminary studies we have found
that RANKL is a weak activator of NF-
B in BMM and RAW 264.7 cells
and that E2 does not decrease the RANKL-induced binding of NF-
B to
its specific consensus sequence (not shown). Moreover, in previous
studies we also found that E2 does not regulate TNF-induced NF-
B
activation (22). Thus, it is likely that the repressive effects of E2
on NF-
B are cell- and promoter-specific.
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FOOTNOTES |
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* This study was supported in part by grants from the National Institutes of Health (AR 41412 and AR 42404), the Eastern Missouri Chapter of the Arthritis Foundation, the Lilly Center for Women's Health, and the Barnes-Jewish Hospital Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These two authors contributed equally to this study.
¶ To whom correspondence should be addressed: Div. of Bone and Mineral Diseases, Barnes-Jewish Hospital, North, 216 S. Kings Highway, St. Louis, MO 63110. Tel.: 314-454-8407; Fax: 314-454-5047; E-mail: Pacifici@imgate.wustl.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010764200
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ABBREVIATIONS |
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The abbreviations used are:
OC, osteoclast;
RANKL, receptor activator of NF-B ligand;
RANK, receptor activator
of NF-
B;
E2, estrogen;
JNK, Jun N-terminal kinase;
M-CSF, macrophage
colony-stimulating factor;
TNF, tumor necrosis factor;
IL, interleukin;
BMM, bone marrow monocyte;
ERK, extracellular signal-related kinase;
MEK, mitogen-activated kinase/extracellular signal-related kinase
kinase;
TRAP, tartrate-resistant acid phosphatase;
PAGE, polyacrylamide
gel electrophoresis.
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