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Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Dr. J. Wesley Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706. E-mail: pike{at}biochem.wisc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone to suppress osteoclast formation induced by
receptor activator of NF-kB ligand (RANKL) and macrophage-colony
stimulating factor in vitro.
5-Dihydrotestosterone suppressed the differentiation of bone marrow
monocytes into osteoclasts from both sham-operated and orchidectomized
mice. Androgen deficiency also led to an increase in the number of
hematopoietic precursors capable of forming osteoclasts and increased
the relative responsiveness of these cells to androgens in
vitro. Interestingly, E2 was as effective as
5-dihydrotestosterone in suppressing osteoclast formation in bone
marrow monocytes from both sham and orchidectomized mice. As with bone
marrow monocytes, 5-dihydrotestosterone also suppressed
RANKL-induced osteoclast formation in the monocyte-macrophagic cell
line RAW264.7. In RAW264.7 cells, androgens appear to block
RANKL-induced osteoclast formation through selective regulation of
c-Jun. Accordingly, 5-dihydrotestosterone suppressed
RANKL-induced c-Jun N-terminal kinase
activation and reduced c-Jun expression levels. These
effects resulted in a reduction in RANKL-induced activator protein-1
DNA binding activity and a corresponding suppression in activator
protein-1-mediated transcriptional activation. These studies
indicate that both E and androgens can suppress osteoclast
formation via a direct, stromal cell-independent action on osteoclast
precursors to block key transcription factors such as
c-Jun essential for osteoclast differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A role for androgens in skeletal regulation is substantiated by numerous studies in humans (8, 9, 10) and rodents (11, 12, 13, 14, 15, 16), demonstrating that chemical or surgical castration, as well as untreated hypogonadism in men, lead to accelerated bone loss. Importantly, the deleterious effects of these conditions on bone can be reversed by treatment with androgens. Despite this, it is unclear whether bone mineral density in males correlates with E or androgen levels (17, 18, 19, 20).
Recent observations of skeletal defects in both ER and aromatase-deficient male rodents and humans suggest the possibility that the active regulatory component in the male skeleton is not T, but rather its aromatizable natural derivative E (19, 20, 21, 22, 23). These studies support a proposal that E plays the dominant role in skeletal homeostasis in both men and women (24); they do not, however, rule out the possibility that androgens also exert actions parallel to those of E. For example, nonaromatizable androgens can protect the human female skeleton from the adverse effects of E deficiency (6). Furthermore, aromatase deficient animals show sex-specific differences in bone mineral density (25), and mice rendered E deficient via treatment with the E antagonist ICI 182,780 show an additional loss of bone mineral density equivalent to ovariectomized animals following treatment with the antiandrogen Casodex (26). Taken together, the above results suggest that both androgens and E exert independent and perhaps overlapping effects to protect bone in males and females, although the relative contributions of each hormone under specific conditions remain to be defined.
ARs have been identified in both osteoblasts (OB) and osteoclasts (OC)
(27), the cells that function to form and degrade bone,
respectively. Accordingly, activation of these receptors by either T or
5-dihydrotestosterone (5-DHT) is capable of modulating a variety
of cellular functions associated with the activity of either OBs or
OCs. For example, T and 5-DHT regulate the expression of certain
genes in OBs that are involved in bone formation (28, 29)
and are also known to inhibit the resorptive capacity of isolated
human, murine, and avian OCs in vitro (30). T
and 5-DHT may also affect OB and OC survival as well (4, 30, 31). Like E, T and 5-DHT also modulate the expression and
secretion of a variety of cytokines and growth factors from OBs and
from AR-positive mesenchymal OB precursors (32), including
macrophage-colony stimulating factor (M-CSF) and the proinflammatory
molecules IL-1, TNF, and IL-6 (26, 33, 34). These factors
(28, 33, 35, 36) as well as the key osteoclast
differentiation-inducing molecule RANKL (37) and its decoy
receptor OPG (12) play regulatory roles in osteoclast
formation, and may form the basis for the communication known to occur
between OBs and OCs during bone remodeling. Their regulation by sex
steroids provides an important mechanism through which supportive cells
exert independent control of osteoclast formation, activity, and
survival.
The fact that AR can be detected in hematopoietic cells with OC potential suggests the possibility that, in addition to the above important activities, androgens may also act to modulate the production of OCs by acting directly on their precursor cells in the marrow. Indeed, we have recently demonstrated that E2 acts directly on primary bone marrow monocytes (BMMs) and monocyte-macrophagic RAW264.7 cells to inhibit soluble RANKL- and M-CSF-induced OC formation in vitro (38). These observations complement our earlier studies that demonstrated that E could inhibit the formation of colony forming units, granulocyte/macrophage (CFU-GM) (39). In the present study, we find that, like E, androgens also suppress RANKL/M-CSF induced osteoclast differentiation/formation from BMMs from both sham and surgically altered [orchidectomized (ORX) or ovariectomized (OVX)] mice and from the murine cell line RAW264.7. We also show that this likely occurs through a suppression of c-Jun N-terminal kinase activity, suppression of c-Jun activation, and down-regulation of c-Jun expression. We hypothesize that these events lead to both a reduction in the number of osteoclast precursors and direct inhibition of RANKL-induced osteoclast formation, both of which are central to the antiresorptive effects of androgens on bone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Dihydrotestosterone (5-DHT) was obtained from Steraloids
(Newport, RI). E2, T, and flutamide (F) were obtained from
Sigma (St. Louis, MO). ICI182780 was obtained from Tocris
Cookson (Ballwin, MO). Murine M-CSF (mM-CSF) was obtained from R&D
Systems (Minneapolis, MN). Recombinant human RANKL (hRANKL) was
expressed as a GST-fusion protein and purified as previously described
(38).
Preparation of murine BMMs
Six-wk-old C57BL/6 sham or ORX male mice and sham or OVX female
mice were obtained from Charles River Laboratories, Inc.
(Wilmington, MA). All animal studies described herein were conducted in
accordance with mandated standards of humane animal care. Bone marrow
cells were isolated from both the tibiae and femurs 2 wk following ORX
or OVX, respectively, and cultured in -MEM with 10% FBS for 24
h. Nonadherent BMMs were isolated and enriched using a Ficoll density
gradient and cultured in phenol-red free -MEM supplemented with 10%
charcoal-stripped FBS. RAW264.7 cells were cultured in phenol-red
free DMEM (low sodium bicarbonate) supplemented with 10%
charcoal-stripped FBS. For stimulation of osteoclast formation, cells
were incubated with 10 ng/ml mM-CSF, soluble hRANKL, or both in the
absence or presence of steroids as indicated.
Characterization and quantitation of osteoclast-like cells
BMMs or RAW264.7 cells were cultured in 48-well plates at a
density of 1 x 105 cells/well or 2 x
103 cells/well, respectively. Cells were treated
with mM-CSF and/or hRANKL as indicated at the beginning of the culture
and during a medium change on d 3 or as otherwise indicated. Cells were
fixed in 10% formaldehyde and stained for tartrate-resistant acid
phosphatase (TRAP) as described previously (38). Briefly,
osteoclast formation was determined by counting the total number of
multinucleated (>3 nuclei), TRAP-positive cells present per well
between d 7 and 10 (BMMs) and d 5 (RAW264.7). Osteoclasts formed from
both cell sources were judged to be authentic through the expression of
numerous additional osteoclast markers including vitronectin receptor,
MMP-9, cathepsin K, carbonic anhydrase II, and calcitonin receptor, and
via their ability to resorb bone in vitro (data not
shown).
RT-PCR analysis
Total RNA was isolated from mouse seminal vesicles and RAW264.7
cells using TRIzol reagent (Life Technologies, Inc.,
Gaithersburg, MD). RNA was reverse transcribed using the Superscript
Preamplification System (Life Technologies, Inc.). The
resulting cDNA was subjected to PCR using previously reported primers
(40). A DNA fragment of 188 bp consistent with mouse AR
was obtained after 30 cycles and visualized using ethidium bromide.
Western blot analysis
RAW264.7 cells were plated at a density of 5 x
106 cells/100 mm dish and treated with hormones
or hRANKL as specified. Following treatment, cell lysates were
subjected to further fractionation and the resultant protein resolved
via SDS-PAGE. Proteins were electrophoretically transferred to
polyvinylidenedifluoride membranes and probed using antibodies specific
to c-Jun, phospho-c-Jun or p38 (New England Biolabs, Inc., Beverly, MA), c-Jun N-terminal kinase
(JNK1) or I
B (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA).
EMSA
Doubled-stranded DNA corresponding to consensus binding sites
for either activating protein (AP)-1 or NF-B were radiolabeled with
32P-
ATP using T4 polynucleototide kinase
(Promega Corp., Madison, WI) and then incubated with
RAW264.7 cell nuclear extracts for 20 min in EMSA binding buffer (5
mM Tris-HCl; 15 mM HEPES buffer, pH 7.9; 100
mM KCl; 3.5 mM MgCl2; 5
mM EDTA; 10% glycerol; 0.1% Tween 20; 5 mM
dithiothreitol; 2 µg poly-deoxyinosine-deoxycytidine per reaction).
Reaction products were resolved on nonreducing 5% polyacrylamide gels,
dried, and autoradiographed.
SAP kinase (JNK1 and p38) assays
Endogenous JNK1 activity was assessed by selectively
immunoprecipitating JNK1 and incubating the precipitate with
radiolabeled 32P-ATP and GST-c-Jun
substrate (residues 1–79) for 30 min as previously described
(38). The substrate was resolved on 4–20% Tris-glycine
gradient gels, and the signals subjected to autoradiography. Endogenous
p38 activity was evaluated using the p38 MAP Kinase assay kit from Cell
Signaling Technology (Beverly, MA), using the manufacturer’s
instructions. The substrate was resolved on 4–20% Tris-glycine
gradient gels, transferred to a membrane, and subjected to Western blot
analysis using an anti-phospho-ATF2 antibody.
Transfections
RAW264.7 cells were seeded into six-well plates at a density of
1 x 106 cells/well. Steroid-treated cells
were preincubated with DHT or E2 as indicated. All cells were
transfected 24 h after plating with luciferase reporter genes as
indicated using Lipofectamine Plus reagent (Life Technologies, Inc.). Plasmids included p36-luc (the control vector containing
the PRL minimal promoter fused to the luciferase gene), p(AP-1)7x-luc
and p(NF-B)3x-luc containing multimerized AP-1 or NF-B response
elements, respectively. After transfection, the media was aspirated and
replaced with DMEM containing 10% charcoal-stripped FBS, in the
presence or absence of steroids and/or RANKL. Cells were harvested
24 h after stimulation and cell lysates assayed for luciferase or
ß-galactosidase activities using standard methods. Luciferase
activity was normalized to ß-galactosidase expression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-DHT to modulate
OC formation in these assays. As seen in Fig. 1A
numerous multinucleated (>3 nuclei),
TRAP-positive OC-like cells resulted when BMMs from either sham or ORX
mice were cultured with RANKL and M-CSF. Interestingly, the number of
OCs formed in BMMs from androgen-deficient animals was approximately
1.6-fold higher than that observed from sham controls. Perhaps more
importantly, exogenously added 5-DHT reproducibly suppressed OC
formation from both SHAM and ORX BMMs (Fig. 1). This effect of 5-DHT
was dose dependent and statistically significant (Fig. 1B, and reversible by the AR antagonist F (Fig. 1A). Surprisingly, the
suppressive effects of 5-DHT were considerably more pronounced in
cells derived from the ORX mice (42% vs. 26% suppression)
(Fig. 1, A and B). These observations, coupled with the increased
number of OCs evident following ORX, suggest either that the OC
precursor population in ORX mice exhibits increased responsiveness to
androgens or that its cellular profile has changed. Nonetheless, the
ability of 5-DHT to suppress OC formation suggests clearly that this
hormone can exert a direct action on OC precursors.
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, both 5-DHT and E2 were
capable of suppressing RANKL- induced OC formation from ORX-derived
BMMs. Moreover, both hormones also exerted a modest suppression of OC
formation in male sham-derived BMMs as well. This cross-sensitivity of
BMMs was confirmed in an analogous experiment with sham and OVX female
mice; BMMs from both sources were equally sensitive to E as well as
androgens (Fig. 6 and data not shown). These observations suggest that
murine BMMs are equally sensitive to both E and androgens regardless of
male or female origin. Perhaps more importantly, like androgen
deficiency, loss of E leads to an elevation in OC precursor cell number
and an increase in apparent sensitivity to both hormones. These studies
support findings that both E and androgens function to protect bone in
males and females (7, 11, 22, 38).
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-DHT or T and quantitated the
number of multinucleated, TRAP-positive cells present after 5 d.
As documented in Fig. 3, RANKL-induced OC
formation was suppressed by both T and 5-DHT (44% and 40%,
respectively). The effects of 5-DHT were statistically significant,
dose dependent (Fig. 3B), and could be completely reversed by adding
the androgen antagonist F (Fig. 3A). These results suggest that like E,
androgens can also suppress RANKL-induced OC formation in RAW264.7
cells. The clonal nature of this line unequivocally supports the
premise that, in addition to their indirect actions on osteoclast
formation through stromal cells, sex steroids can also exert direct
effects on precursor cells to suppress OC formation.
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-DHT to suppress OC formation in both BMMs and
RAW264.7 cells at nanomolar concentrations and the capacity of F to
reverse this inhibition are suggestive of an AR-mediated action. Thus,
total RNA isolated from both RAW264.7 cells and from mouse seminal
vesicles was reverse transcribed and subjected to PCR analysis using
primers specific for mouse AR. As expected, PCR analysis revealed the
presence of transcripts for AR from both tissue sources, as assessed by
generation of the appropriately sized DNA fragments (Fig. 3C
, 188 bp).
Preliminary experiments using DNA cellulose affinity chromatography
coupled to Western blot analysis suggest that AR protein is present in
these cells, although its level, like that of ER and
ß(38), is clearly very low (data not shown). Regardless,
these results together with the biological effects of 5-DHT and F on
RAW264.7 cells support an AR-mediated mechanism.
Androgens and E suppress OC formation via specific activation of
their cognate receptors
In view of the fact that both AR and ER are likely expressed in
both BMMs and RAW264.7 cells, we next employed the receptor selective
antagonists ICI-182780 (ICI) and F to confirm that the activity of each
hormone was mediated specifically via activation of its cognate nuclear
receptor. Although both 5-DHT and E2 suppressed RANKL-induced OC
differentiation in RAW264.7 cells, the action of each of the hormones
was reversible only by its respective antagonist (Fig. 4). Although significant in other
experiments (Figs. 1 and 3), reversal of 5-DHT mediated suppression
by F was not statistically significant at P < 0.05 in
the experiment illustrated, although the trend toward reversal was
evident. Importantly, this antagonist selectivity also extended to BMMs
from OVX mice, where identical results were obtained (Fig. 4). These
results support that idea that E and androgens suppress RANKL-induced
osteoclastogenesis in both BMMs and RAW264.7 cells, and that these
effects are specific and mediated selectively via activation of each
hormone’s specific cognate receptor.
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-DHT. To determine whether the
actions of 5-DHT occur early in the process of OCs formation, we
treated RAW264.7 cells with M-CSF and RANKL, added 5-DHT on d 0, 3,
or 5 and assessed OC formation on d 7. The number of OCs formed as a
function of this treatment regime was compared with those obtained when
5-DHT was added at multiple time points (d 0, 3, and 5). As is
evident in Fig. 5, a single dose of
5-DHT on d 0 was as effective in suppressing RANKL-induced OC
formation as multiple treatments throughout the entire 5-d period.
Treatment on d 3 or 5 was also effective in inhibiting
osteoclastogenesis, although to a lesser degree. Although these studies
do not rule out actions of 5-DHT on OC formation or survival at a
later stage, they do suggest that the hormone can function early in the
process of OC formation, perhaps during the initial stages of
differentiation.
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-DHT might block early RANKL signaling events
which have been shown to be critical to OC formation. RANKL interaction
with its receptor RANK initiates activation of several distinct
intracellular signaling pathways whose components include the SAPKs,
the MAPKs, the c-src/PI3K/PKB pathway and NF-B (41, 42). Because activation of ERK1/2 and c-PKB activities appear to
be involved predominantly in OC survival, we focused on the ability of
5-DHT to suppress the RANKL-induced activity of the two SAPKs JNK1
and p38 in RAW264.7 cells. 5-DHT suppressed RANKL-induced JNK1
activity in a F-reversible manner in the absence of any change in JNK1
protein levels (Fig. 6A). This
suppression was time dependent, however, requiring the presence of
5-DHT for at least 8 h before RANKL stimulation. In contrast,
neither 5-DHT nor E2 was able to suppress RANKL-induced p38 kinase
activity (Fig. 6B). Because previous observations indicate that E2 also
suppresses JNK1 activity (38, 44), these data indicate
that both hormones may function to selectively modulate the activity of
this enzyme.
NF-B activation in RAW264.7 cells is not inhibited by
androgens
In addition to its actions on JNK1 and p38 kinase, we also
examined the ability of 5-DHT to suppress RANKL-induced NF-B
activation. The importance of NF-B in the process of osteoclast
formation has been clearly demonstrated in p50/p52 null mutant mice
which exhibit a significant impairment in the ability to form OCs
(43). As observed in Fig. 7A, pretreatment of RAW264.7 cells with
either 5-DHT (or E2) 24 h before RANKL stimulation failed to
prevent RANKL-induced binding of NF-B to a consensus NF-B
response element as assessed by EMSA. These two hormones also failed to
suppress NF-B DNA binding when added together with RANKL (data not
shown). The failure of 5-DHT (and E2) to suppress NF-B activation
was also confirmed through analysis of IB degradation wherein
5-DHT failed to inhibit RANKL-induced IB degradation (Fig. 7B).
Finally, and perhaps most importantly, neither 5-DHT nor E2 were
capable of suppressing the RANKL-induced transcriptional activity of an
NF-B regulated luciferase reporter gene. The inability of both
hormones to suppress either p38 kinase activity or RANKL-induced
NF-B activation indicates that hormonal suppression is pathway
specific.
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-DHT can selectively
down-regulate the activity of JNK1, an observation consistent with
previous studies using E2 (38, 44). c-Jun
represents a major target of JNK1 and is an important heterodimeric
partner of c-Fos. Thus, a potential consequence of the down-regulation
of this enzyme could be the inhibition of c-Jun
transcriptional activation and AP-1 activity. We first examined whether
5-DHT could block RANKL-induced c-Jun phosphorylation or
alter c-Jun expression in RAW264.7 cells. Treatment of
RAW264.7 cells with RANKL for periods up to 2 h revealed a
transient phosphorylation of c-Jun that peaked at 30 min as
well as a steady accumulation of c-Jun protein that also
peaked at 30 min (Fig. 8, left
panel). Pretreatment of the cells with 5-DHT for 16 h
caused a dramatic reduction in relative levels of c-Jun and
a delay in the rate of phosphorylation such that the peak was observed
at 60 rather than at 30 min (Fig. 8, right panel). Despite
these findings, treatment with RANKL induced the expression of
c-Jun to the extent that cellular production of this protein
was restored to vehicle-treated levels within 60 min.
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-DHT on c-Jun expression above prompted us to examine
whether this transient effect of the hormone could result in a change
both in AP-1 DNA binding and in transcriptional activity. RAW264.7
cells were pretreated with either vehicle, 5-DHT, or E2 for 16
h, stimulated for 30 min with RANKL and then assayed for the capacity
of nuclear protein extracts to bind to an AP-1 consensus
oligonucleotide. As observed in Fig. 9A, treatment with RANKL led to a substantial increase in AP-1 binding to
DNA. Antibodies to c-Jun retarded migration of the complex,
indicating that c-Jun was a predominant component of the
complex (data not shown). Treatment with 5-DHT or E2, however,
completely abrogated RANKL-induced AP-1 binding; neither hormone
affected the AP-1 binding activity of unstimulated cells. Importantly,
this finding correlated with the subsequent observation that 5-DHT
and E2 also suppressed RANKL-induced activation of an AP-1-dependent
reporter gene following transfection into RAW264.7 cells (approximately
32% and 45% suppression, respectively) (Fig. 9B). These studies
suggest that despite the transient nature of 5-DHT’s action of JNK1
activity and c-Jun expression seen in Fig. 8, these effects
lead to a functional consequence in RANKL signaling.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-DHT
suppresses the activity or expression of key components within the RANK
signaling pathway that lead to OC formation, namely the SAPK JNK1 and
its transcription factor target c-Jun. This action appears
to be specific because 5-DHT fails to suppress the SAPK p38 and does
not suppress RANKL-induced activation of NF-B. Although suppression
of these components is transient, requiring pretreatment with the
hormone for at least 8 h, additional studies suggest that 5-DHT
also reduces AP-1 DNA binding and AP-1-dependent transactivation. Early
events in RANKL signaling are presently linked only circumstantially to
OC differentiation. Nevertheless, we hypothesize that they are integral
to subsequent suppression of the monocyte-macrophage phenotype and
induction of the OC phenotype. As such, suppression of these events by
both androgens and E represents a novel mechanism through which the
hormones can modulate OC formation and thus bone resorption. Loss of either androgens or E leads to an up-regulation of OC precursor number and an apparent increase in responsiveness to both hormones. These results indicate that E deficiency in females and androgen deficiency in males specifies a common physiological process that results in increased OC precursor number. It should be noted here that these studies do not define androgens as the direct regulators of osteoclast precursor number, because the depletion in circulating levels of androgens that results from ORX would likely result in reduction in E2 levels as well. Thus, one explanation for the similarity in response of OC precursors to ORX and OVX may well be that the active common ligand in both cases is E, as proposed earlier (21, 24). Regardless, the nature of this elevated precursor population and its increased sensitivity to both E and androgens following either ORX or OVX is particularly interesting. These data, together with the finding that neither hormone can suppress osteoclast formation substantially below the sham control level of osteoclast production, suggest that two subpopulations of osteoclast precursors may be present—those that respond to hormones and those that do not. Identifying such populations will require the ability to sort these diverse groups of cells using selected myeloid and OC precursor markers.
An alternative explanation for the similarity of response to both E and
androgens is that a common cellular mechanism mediates the actions of
both hormones in bone cells. Considerable evidence suggests that E can
function via a membrane receptor(s) to initiate signaling pathways such
as that of the MAPKs in a variety of cell types (45). This
evidence is supported by findings that suggest that cell impermeable
BSA conjugates of E2 can activate some of these pathways
(46). Other proposals suggest that receptors for both
hormones interact in certain tissues and thus mediate a common
downstream effect regardless of the identity of the ligand
(47). Finally, recent studies in OB cells suggests a
common unisex mechanism that appears to be neither specific for E or
androgens nor selective for the receptor-specific antagonists ICI
182780 and F yet requires the presence of ER or AR (48).
Our studies support a traditional intracellular receptor mechanism that
exhibits classical hormonal and receptor selectivity and appears to
involve transcriptional regulation. The presence of transcripts for
both ER and AR in marrow cells and RAW264.7 cells supports this
mechanism, as does the time required for activation. Additional
evidence for a traditional hormone response resides in the ability of F
and ICI182780 to block selectively 5-DHT and E2 action,
respectively, in both BMMs and RAW264.7 cells and to not act as cross
antagonists of AR and ER activity, respectively (49).
These characteristics argue for a traditional mechanism of action of
5-DHT and E in these bone cells.
How does 5-DHT (and E) suppress OC formation? Our data suggest that
androgens may act early in the process of osteoclastogenesis and might
function to suppress OC differentiation by selectively blocking certain
RANKL activated signaling components essential to OC formation such as
JNK1 and c-Jun. A direct link between early RANKL-induced
cellular events and the subsequent formation of OCs has yet to be
defined, however. As a result, it remains difficult to connect the
actions of 5-DHT (and E) on these early events to the production of
OCs 5 d later. The mechanism through which 5-DHT and E suppress
JNK1 activity also remains to be identified. We have ruled out the
possibility that RANK expression is simply down-regulated by hormone
for two reasons: 1) there is no apparent change in the expression of
RANK transcripts in response to hormone (38); and 2) a
reduction in this receptor’s level would likely cause not only an
inhibition of JNK1 activity but also a blockade of p38 and NF-B
activity as well. Further studies will be required to determine how
each of these hormones’ suppresses c-Jun gene
expression.
In summary, we have shown that both T and 5-DHT suppress RANKL and
M-CSF-induced OC formation from both BMM and RAW264.7 cells. This
reduction is mediated through suppression of JNK1 activity and
down-regulation of c-Jun expression, both of which lead to
an apparent suppression of AP-1 functional activity essential to
osteoclast formation. Although the regulation of the c-Jun
pathway by sex hormones appears to be specific, it may represent only
one of several mechanisms through which sex steroids suppress OC
formation. Thus, future studies will focus on defining molecular
mechanisms whereby modulation of these and other regulatory factors
leads to down-regulation of OC formation.
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Acknowledgments |
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Footnotes |
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Abbreviations: AP-1, Activator protein-1; BMM, bone
marrow monocytes; CFU-GM, colony forming units; 5-DHT;
5-dihydrotestosterone; F, flutamide; JNK1, c-Jun
N-terminal kinase; M-CSF, macrophage colony-stimulating factor; OB,
osteoblast; OC, osteoclast; ORX, orchidectomized; OVX, ovariectomized;
hRANKL, recombinant human RANKL; SAPK, stress-activated protein kinase;
TRAP, tartrate-resistant acid phosphatase.
Received April 10, 2001.
Accepted for publication May 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Dihydrotestosterone partially restores cancellous bone volume in
osteopenic ovariectomized rats. Am J Physiol 267:E853–E859
- and ß-isoforms, and of the androgen
receptor in cultures of native mouse bone marrow and of
stromal/osteoblastic cells. Bone 24:465–473[Medline]
B by RANK requires tumor necrosis factor receptor-
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of a novel TRAF6 interaction motif. J Biol Chem 274:7724–7731B (RANK). Interaction with tumor necrosis factor
receptor-associated factors and activation of NF-B and c-Jun
N-terminal kinase. J Biol Chem 273:20551–20555B leads to multifocal
defects in immune responses. Cell 80:321–330[CrossRef][Medline]
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