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Endocrinology Vol. 142, No. 9 3800-3808
Copyright © 2001 by The Endocrine Society

ARTICLES

Androgens Suppress Osteoclast Formation Induced by RANKL and Macrophage-Colony Stimulating Factor

Dustin M. Huber, Amy C. Bendixen, Peterson Pathrose, Sunil Srivastava, Krista M. Dienger, Nirupama K. Shevde and J. Wesley Pike

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgen deficiency in males leads to an increase in osteoclastic bone resorption and a progressive decrease in bone mineral density. In the current studies, we examined the ability of 5{alpha}-dihydrotestosterone to suppress osteoclast formation induced by receptor activator of NF-kB ligand (RANKL) and macrophage-colony stimulating factor in vitro. 5{alpha}-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{alpha}-dihydrotestosterone in suppressing osteoclast formation in bone marrow monocytes from both sham and orchidectomized mice. As with bone marrow monocytes, 5{alpha}-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{alpha}-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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOPOROSIS IS A disease in which skeletal integrity is compromised due to decreased bone mass, leading to a significantly enhanced risk of skeletal fracture (1). The exact cellular mechanisms underlying the pathogenesis of osteoporosis have yet to be identified. Bone loss, however, most often occurs as a result of an imbalance in the normal bone remodeling process that favors bone resorption and degradation over bone formation (1, 2, 3, 4). Two of the most significant determinants of progressive bone loss and osteoporosis in both males and females are age and sex hormone status (5, 6, 7). Accordingly, both postmenopausal women as well as aging men experience increased bone turnover, decreased bone mineral density and increased risk of fracture.

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{alpha}-dihydrotestosterone (5{alpha}-DHT) is capable of modulating a variety of cellular functions associated with the activity of either OBs or OCs. For example, T and 5{alpha}-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{alpha}-DHT may also affect OB and OC survival as well (4, 30, 31). Like E, T and 5{alpha}-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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
5{alpha}-Dihydrotestosterone (5{alpha}-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 {alpha}-MEM with 10% FBS for 24 h. Nonadherent BMMs were isolated and enriched using a Ficoll density gradient and cultured in phenol-red free {alpha}-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{kappa}B{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

EMSA
Doubled-stranded DNA corresponding to consensus binding sites for either activating protein (AP)-1 or NF-{kappa}B were radiolabeled with 32P-{gamma}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-{gamma}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-{kappa}B)3x-luc containing multimerized AP-1 or NF-{kappa}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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OC formation from BMM precursors is enhanced by ORX and inhibited by exogenous androgens
To examine the potential regulatory role of androgens in OC formation, we cultured BMMs from androgen-normal sham-operated (sham) and androgen-deficient ORX male mice in the presence of RANKL and M-CSF and quantitated the number of OCs that formed after 10 d. We further assessed the ability of exogenously added 5{alpha}-DHT to modulate OC formation in these assays. As seen in Fig. 1AGo 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{alpha}-DHT reproducibly suppressed OC formation from both SHAM and ORX BMMs (Fig. 1Go). This effect of 5{alpha}-DHT was dose dependent and statistically significant (Fig. 1BGo, and reversible by the AR antagonist F (Fig. 1AGo). Surprisingly, the suppressive effects of 5{alpha}-DHT were considerably more pronounced in cells derived from the ORX mice (42% vs. 26% suppression) (Fig. 1Go, 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{alpha}-DHT to suppress OC formation suggests clearly that this hormone can exert a direct action on OC precursors.



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  		Figure 1. Androgens inhibit OC formation in primary murine monocytes in the absence of stroma. A, BMMs from SHAM (left panel) or ORX (right panel) mice were stimulated with mM-CSF (10 ng/ml) and shRANKL (2 nM) in the presence of vehicle, 5{alpha}-DHT (10-8 M), 5{alpha}-DHT (10-8 M) and F (10-6 M) or F (10-6 M) alone and OC number quantitated on d 7. B, BMMs from SHAM (left panel) or ORX (right panel) mice were stimulated with mM-CSF (10 ng/ml) and RANKL (2 nM) in the presence of increasing concentrations of 5{alpha}-DHT and OC number similarly quantitated. Values represent the mean ± SE, n = 3 (b and c are significant vs. a at P < 0.05; d, e,and f are significant vs. c at P < 0.05, ANOVA) and are representative of three separate experiments.

 
E suppresses BMM-dependent OC formation from normal and androgen-deficient (ORX) male mice
The results of the previous experiments suggest that androgen deficiency induced by ORX leads to an increase in OC precursor number and/or an increase in responsiveness to androgens, findings identical with that observed following OVX (38). In view of this, we next assessed whether suppression of OC formation following ORX was specific for androgens or could be mimicked by exogenously added E2. As seen in Fig. 2Go, both 5{alpha}-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. 6Go 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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  		Figure 2. E inhibits OC formation in BMMs from ORX mice. BMMs were isolated from either SHAM (left panel) or ORX (right panel) mice and treated with mM-CSF (10 ng/ml) and RANKL (2 nM) in the presence of vehicle, 5{alpha}-DHT (10-8 M), or E2 (10-8 M) and OCs quantitated on d 7. Values represent the mean ± SE, n = 3 (b is significant vs. a at P < 0.05, c is significant vs. a, and d is significant vs. c at P < 0.001, ANOVA) and are representative of three separate experiments.

 


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  		Figure 6. 5{alpha}-DHT inhibits JNK1 but not p38 kinase activity in RAW264.7 cells. A, RAW264.7 cells (106 cells/ml) were pretreated with either vehicle or 5{alpha}-DHT (10-8 M) for periods up to 24 h or for 24 h with either F (10-6 M) or 5{alpha}-DHT (10-8 M) and F (10-6 M) as indicated, stimulated for 15 min with RANKL (5 nM) and then used to prepare nuclear extracts. JNK1 activity was assessed following immunoprecipitation using GST-c-Jun (residues 1–79) as a substrate as described in Materials and Methods (upper panel). Total JNK1 protein was evaluated using Western blot analysis (lower panel). These results are representative of six separate experiments. B, RAW264.7 cells were pretreated as in (A) with either vehicle, 5{alpha}-DHT, E2 (10-8 M), or F (10-6 M), stimulated for 15 min with RANKL (5 nM) and then used to prepare nuclear extracts. Immunoprecipitated total p38 kinase activity was assessed following immunoprecipitation using a GST-ATF2 fragment as substrate and the product resolved by SDS-PAGE. Phospho-ATF2 was detected using Western blot analysis with an anti-phospho-ATF2 antibody (upper panel). Total p38 protein was detected using Western blot analysis (lower panel). These results are representative of three separate experiments.

 
Androgens inhibit OC formation in the murine monocytic cell line RAW264.7
BMM preparations can be contaminated with stromal cells. To rule out the contribution of stromal cells definitively in these assays, we employed murine RAW264.7 cells. Previous studies have demonstrated that these cells can be induced to form authentic osteoclasts in vitro by RANKL, and that this induction is suppressed by E2 (38). In light of the latter observation, we tested the possibility that androgens might also suppress osteoclast formation in this cell line. We therefore treated RAW264.7 cells with RANKL and M-CSF in the absence or presence of 5{alpha}-DHT or T and quantitated the number of multinucleated, TRAP-positive cells present after 5 d. As documented in Fig. 3Go, RANKL-induced OC formation was suppressed by both T and 5{alpha}-DHT (44% and 40%, respectively). The effects of 5{alpha}-DHT were statistically significant, dose dependent (Fig. 3BGo), and could be completely reversed by adding the androgen antagonist F (Fig. 3AGo). 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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  		Figure 3. Androgens inhibit OC formation in the murine monocytic cell line RAW264.7. A, RAW264.7 cells were treated with mM-CSF (10 ng/ml) and RANKL (2 nM) in the presence of vehicle, T (10-8 M), 5{alpha}-DHT (10-8 M), or 5{alpha}-DHT (10-8 M) and F (10-6 M) and OCs quantitated on d 5. Values represent the mean ± SE, n = 3 (b is significant vs. a at P < 0.005, ANOVA) and are representative of three independent experiments. B, RAW264.7 cells were treated with mM-CSF (10 ng/ml) and RANKL (2 nM) in the presence of increasing concentrations of 5{alpha}-DHT and OC number determined as in (A). Values represent the mean ± SE, n = 3 (b, c and d are significant vs. a at P < 0.005, ANOVA) and are representative of three separate experiments. C, Total RNA from both RAW264.7 cells and seminal vesicle tissue was isolated and subjected to RT-PCR analysis using primers to murine AR. The 188 bp amplicon detected from both sources corresponds to the expected fragment from the AR transcript. Primers to GAPDH were used as an amplification control.

 
RAW264.7 cells express AR transcripts
The ability of 5{alpha}-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. 3CGo, 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 {alpha} and ß(38), is clearly very low (data not shown). Regardless, these results together with the biological effects of 5{alpha}-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{alpha}-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. 4Go). Although significant in other experiments (Figs. 1Go and 3Go), reversal of 5{alpha}-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. 4Go). 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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  		Figure 4. Androgens and E inhibit OC formation in BMMs from OVX mice or from RAW264.7 cells via their cognate receptors. BMMs from OVX mice or RAW264.7 cells were stimulated with mM-CSF (10 ng/ml) and RANKL (2 nM) in the presence of the indicated combination of 5{alpha}-DHT (10-8 M), E2 (10-8 M), F (10-6 M), ICI182780 (10-6 M) (ICI) and OC formation assessed on d 7 (BMMs) or on d 5 (RAW264.7 cells). Values represent the mean ± SE, n = 3 (b, c are significant vs. a; g, h are significant vs. f; e is significant vs. c; j is significant vs. h at P < 0.05, ANOVA) (d is significant vs. b at P < 0.10, ANOVA) (i is significant vs. g at P < 0.065, ANOVA) and are representative of at least two identical experiments using BMMs and three experiments using RAW264.7 cells.

 
Inhibition of OC formation in RAW264.7 cells by androgens occurs early in OC differentiation
Osteoclastogenesis is a complex process encompassing cellular proliferation, differentiation, fusion, activation and survival (2); this process requires at least 5–6 d in RAW264.7 cells. As a consequence, each of these cellular events is a potential target process for regulation by 5{alpha}-DHT. To determine whether the actions of 5{alpha}-DHT occur early in the process of OCs formation, we treated RAW264.7 cells with M-CSF and RANKL, added 5{alpha}-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{alpha}-DHT was added at multiple time points (d 0, 3, and 5). As is evident in Fig. 5Go, a single dose of 5{alpha}-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{alpha}-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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  		Figure 5. Inhibition of OC formation in RAW264.7 cells by 5{alpha}-DHT occurs early in the process of OC differentiation. RAW264.7 cells were treated with mM-CSF (10 ng/ml) and RANKL (2 nM) with 5{alpha}-DHT (10-8 M) added at the indicated times. Values represent the mean ± SE, n = 3 (b, c, and d are significant vs. a at P < 0.001, ANOVA) and are representative of two identical experiments.

 
Androgens selectively inhibit JNK1 but not p38 stress-activated protein kinase (SAPK) activity in RAW264.7 cells
Because RANKL is largely a differentiation factor, we explored the possibility that 5{alpha}-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-{kappa}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{alpha}-DHT to suppress the RANKL-induced activity of the two SAPKs JNK1 and p38 in RAW264.7 cells. 5{alpha}-DHT suppressed RANKL-induced JNK1 activity in a F-reversible manner in the absence of any change in JNK1 protein levels (Fig. 6AGo). This suppression was time dependent, however, requiring the presence of 5{alpha}-DHT for at least 8 h before RANKL stimulation. In contrast, neither 5{alpha}-DHT nor E2 was able to suppress RANKL-induced p38 kinase activity (Fig. 6BGo). 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-{kappa}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{alpha}-DHT to suppress RANKL-induced NF-{kappa}B activation. The importance of NF-{kappa}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. 7AGo, pretreatment of RAW264.7 cells with either 5{alpha}-DHT (or E2) 24 h before RANKL stimulation failed to prevent RANKL-induced binding of NF-{kappa}B to a consensus NF-{kappa}B response element as assessed by EMSA. These two hormones also failed to suppress NF-{kappa}B DNA binding when added together with RANKL (data not shown). The failure of 5{alpha}-DHT (and E2) to suppress NF-{kappa}B activation was also confirmed through analysis of I{kappa}B degradation wherein 5{alpha}-DHT failed to inhibit RANKL-induced I{kappa}B degradation (Fig. 7BGo). Finally, and perhaps most importantly, neither 5{alpha}-DHT nor E2 were capable of suppressing the RANKL-induced transcriptional activity of an NF-{kappa}B regulated luciferase reporter gene. The inability of both hormones to suppress either p38 kinase activity or RANKL-induced NF-{kappa}B activation indicates that hormonal suppression is pathway specific.



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  		Figure 7. Androgens do not inhibit RANKL induced NF-{kappa}B DNA binding or activity. A, RAW264.7 cells (106 cells/ml) were pretreated with either vehicle, 5{alpha}-DHT (10-8 M) or E2 (10-8 M) for 16 h and then stimulated with either vehicle or RANKL (5 nM) for 30 min. High-salt nuclear extracts (10 µg protein) were incubated with a radiolabeled NF-{kappa}B consensus sequence for 20 min and then resolved as described in Materials and Methods. The free probe, the NF-{kappa}B complex, and a nonspecific complex are indicated by arrows. B, RAW264.7 cells (106 cells/ml) were pretreated with either vehicle, 5{alpha}-DHT (10-8 M) or E2 (10-8 M) for 16 h and then stimulated with either vehicle or RANKL (5 nM) for 15 min. High-salt nuclear extracts (75 µg protein) were resolved using SDS-PAGE, transferred to a polyvinylidendifluoride membrane and probed for I{kappa}B{alpha} by Western blot analysis. I{kappa}B{alpha} is indicated by the arrow. C, RAW264.7 cells (106 cells/ml) were transfected with p(NF-{kappa}B)3x-luc and then treated with either vehicle, RANKL (5 nM), or RANKL (5 nM) plus either 5{alpha}-DHT (10-8 M) or E2 (10-8 M). Luciferase activity was assessed 24 h later and normalized using ß-galactosidase activity. A 16 h pretreatment of RAW264.7 cells with either hormone had no effect on RANKL-induced p(NF-{kappa}B)3x-luc activity (data not shown). RANKL, 5{alpha}-DHT or E2 had no effect on the activity of p36-Luc (control vector) following transfection into RAW264.7 cells (data not shown). Values represent the mean ± SE, n = 3 and are representative of three similar experiments.

 
Androgens suppress c-Jun protein levels in RAW 264.7 cells
The aforementioned results suggest that 5{alpha}-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{alpha}-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. 8Go, left panel). Pretreatment of the cells with 5{alpha}-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. 8Go, 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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  		Figure 8. Androgens down-regulate c-Jun expression in RAW264.7 cells. RAW264.7 cells (106/ml) were treated with either vehicle or 5{alpha}-DHT (10-8 M) for 16 h and then stimulated with RANKL (5 nM) for the indicated times. High-salt nuclear extracts (75 µg protein) were resolved using SDS-PAGE and then subjected to Western blot analysis using anti-phospho c-Jun (upper panel) or total c-Jun (lower panel) antibodies as indicated in Materials and Methods. The results are representative of five similar experiments.

 
Sex steroids inhibit AP-1 DNA binding and suppress transactivation in vitro
The ability of RANKL to reverse the suppressive effects of 5{alpha}-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{alpha}-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. 9AGo, 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{alpha}-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{alpha}-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. 9BGo). These studies suggest that despite the transient nature of 5{alpha}-DHT’s action of JNK1 activity and c-Jun expression seen in Fig. 8Go, these effects lead to a functional consequence in RANKL signaling.



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  		Figure 9. Androgens down-regulate AP-1 DNA binding and inhibit AP-1-dependent transcription in vitro. A, RAW264.7 cells (106 cells/ml) were pretreated with either vehicle, 5{alpha}-DHT (10-8 M) or E2 (10-8 M) for 16 h and then stimulated with either vehicle or RANKL (5 nM) for 30 min. High-salt nuclear extracts (10 µg protein) were incubated with a radiolabeled AP-1 consensus duplex oligonucleotide for 20 min and then resolved as described in Materials and Methods. Migration of the free probe and the AP-1/DNA complex are indicated with arrows. Data are representative of two similar experiments. B, RAW264.7 cells (106 cells/ml) were transfected with p(AP-1)7x-luc and then treated with either vehicle, RANKL (5 nM), or RANKL (5 nM) plus either 5{alpha}-DHT (10-8 M) or E2 (10-8 M). Luciferase activity was assessed 24 h later and normalized using ß-galactosidase activity. Pretreatment of cells with either hormone enhanced the suppressive effects of both 5{alpha}-DHT and E2 on p(AP-1)7x-luc (data not shown). Values represent the mean ± SE, n = 3 (b vs. a at P < 0.001, ANOVA) and are representative of three similar experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies indicate that androgens suppress the formation of OCs at least in part through an action independent of stromal cells. These actions are dose dependent, inhibited by F and occur at concentrations reflective of an AR-mediated action. We also show that 5{alpha}-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{alpha}-DHT fails to suppress the SAPK p38 and does not suppress RANKL-induced activation of NF-{kappa}B. Although suppression of these components is transient, requiring pretreatment with the hormone for at least 8 h, additional studies suggest that 5{alpha}-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{alpha}-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{alpha}-DHT and E in these bone cells.

How does 5{alpha}-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{alpha}-DHT (and E) on these early events to the production of OCs 5 d later. The mechanism through which 5{alpha}-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-{kappa}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{alpha}-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.


   Acknowledgments
 
We thank Glenn Doerman for his contributions to this manuscript and Dr. Chris K. Glass for providing luciferase expression plasmids.


   Footnotes
 
This work was supported in part by NIH Grant DK-56059.

Abbreviations: AP-1, Activator protein-1; BMM, bone marrow monocytes; CFU-GM, colony forming units; 5{alpha}-DHT; 5{alpha}-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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 Discussion
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