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Endocrinology, doi:10.1210/en.2005-0717
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Endocrinology Vol. 147, No. 7 3613-3622
Copyright © 2006 by The Endocrine Society

Glucocorticoid Regulation of Osteoclast Differentiation and Expression of Receptor Activator of Nuclear Factor-{kappa}B (NF-{kappa}B) Ligand, Osteoprotegerin, and Receptor Activator of NF-{kappa}B in Mouse Calvarial Bones

Charlotte Swanson, Mattias Lorentzon, H. Herschel Conaway and Ulf H. Lerner

Department of Oral Cell Biology (C.S., H.H.C., U.H.L.), Umeå University, SE-901 87 Umeå, Sweden; Center for Bone Research at the Sahlgrenska Academy (C.S., M.L.), Department of Internal Medicine, Göteborg University, SE-413 45 Göteborg, Sweden; and Department of Physiology and Biophysics (H.H.C.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Professor Ulf H. Lerner, D.D.S., Ph.D., Department of Oral Cell Biology, Umeå University, Umeå SE-901 87, Sweden. E-mail: ulf.lerner{at}odont.umu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, dexamethasone treatment of neonatal mouse calvarial bones increased mRNA expression of tartrate-resistant acid phosphatase, calcitonin receptor (CTR), cathepsin K, carbonic anhydrase II, osteoprotegerin (OPG), and receptor activator of nuclear factor-{kappa}B (RANK) as well as mRNA and protein expression of RANK ligand (RANKL). The increase in OPG mRNA noted with dexamethasone was in contrast to 1,25(OH)2-vitamin D3 (D3) treatment, which decreased OPG expression. Stimulation of 45Ca release by dexamethasone and hydrocortisone in calvariae was blocked by OPG. Stimulation of RANKL, RANK, OPG, and CTR mRNA expression by dexamethasone in calvariae was blocked by the glucocorticoid receptor antagonist RU 38 486. Greater than additive potentiations of CTR mRNA and RANKL mRNA and protein were observed when D3 and dexamethasone were combined. Vitamin D receptor mRNA was increased by dexamethasone and D3, whereas glucocorticoid receptor (GR) mRNA was decreased by dexamethasone and unaffected by D3. No synergistic interaction between dexamethasone and D3 on either vitamin D receptor or GR mRNA expression was noted. The data demonstrate that dexamethasone-induced bone resorption in calvarial bones is associated with increased differentiation of osteoclasts and regulation of the RANKL-RANK-OPG system. The increase in OPG expression and the decrease of GR expression noted with dexamethasone offer an explanation for why bone breakdown in mouse calvariae treated with glucocorticoids is less than that caused by resorptive agents like D3. The synergistic stimulation of RANKL by dexamethasone and D3 offers an explanation of how glucocorticoids and D3 interact to potentiate bone resorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TREATMENT WITH GLUCOCORTICOIDS often results in a substantial loss of bone that is accompanied by fracture (1, 2, 3, 4, 5, 6, 7, 8). Bone loss caused by glucocorticoids is characterized by an early phase of increased bone resorption followed by a later phase of decreased bone formation (9). Previous explanations for the increase in bone breakdown caused by glucocorticoids had suggested that this effect might be indirect and due to increased PTH secretion (10, 11, 12). However, recent in vitro studies indicated that glucocorticoids can act directly on bone cells to stimulate osteoclast formation and resorption (13, 14, 15, 16, 17, 18, 19). In bone marrow cultures and cocultures of stromal cells and spleen cells stimulated with 1,25(OH)2-vitamin D3 (D3), cotreatment with glucocorticoids increases the number of osteoclasts formed and enhances their activity (17, 18). In organ-cultured fetal rat parietal bones (13) and neonatal mouse calvariae (14, 15), glucocorticoid exposure increases bone resorption. When glucocorticoids are combined with D3 in neonatal mouse calvariae, synergistic potentiation of bone resorption occurs (16).

Although they are good stimulators of osteoclast formation and activity, glucocorticoids also have been shown to decrease the survival of terminally differentiated, multinucleated osteoclasts (20, 21). This observation may help explain why the amount of bone resorbed by glucocorticoids in vitro is less than that seen after stimulation with compounds like D3 or PTH.

Three recently discovered molecules in the TNF ligand and receptor superfamilies (reviewed in Refs.22, 23, 24): receptor activator of nuclear factor-{kappa}B ligand (RANKL), osteoprotegerin (OPG), and receptor activator of nuclear factor-{kappa}B (RANK) are thought to play important roles in osteoclastogenesis. RANKL exists as both a membrane protein in stromal cells/osteoblasts and a soluble protein. Expression of RANKL is increased by numerous stimulators of bone resorption, including well-known agents like PTH and D3. RANKL activates the receptor RANK, which is found on the surface of osteoclast progenitor cells and terminally differentiated, multinucleated osteoclasts. The interaction between RANKL and RANK can be inhibited by OPG, a soluble decoy receptor released from stromal cells/osteoblasts. Gene deletion studies have emphasized the important role of the RANKL-RANK-OPG system in osteoclastogenesis. Mice lacking RANKL or RANK have an osteopetrotic phenotype with very few osteoclasts (25, 26), whereas deletion of the OPG gene results in early onset osteoporosis with an abundance of osteoclasts (27).

Many stimulators of bone resorption are thought to increase RANKL expression and decrease OPG expression. In the case of glucocorticoids, it has been reported that these steroids inhibit OPG mRNA and protein expression in isolated human osteoblast-like cells, human bone marrow stromal cells, immortalized human marrow stromal cells, immortalized human fetal osteoblastic cells, and the osteoblastic cell line MG-63 (28, 29, 30, 31). Decreased circulating OPG levels have also been found in patients receiving glucocorticoid treatment (32, 33). Fewer studies have been performed on the effect of glucocorticoids on RANKL expression. It has been reported that dexamethasone does not have an effect on RANKL mRNA in murine bone marrow ST2 cells (34). However, increased RANKL mRNA expression has been observed in immortalized human fetal osteoblastic cells and human bone marrow stromal cells (28), MG-63 cells (31), and primary human osteoblasts (19) after exposure to dexamethasone.

There have been numerous studies on RANKL and OPG regulation in skeletal tissue, but the number of investigations that have focused on RANK is small. Increased RANK mRNA and protein levels have been observed in the murine monocytic cell line RAW 264.7 after treatment with TGF-ß (35), and an increase in RANK mRNA has been noted in calvarial bones after treatment with D3 (36). The number of reports demonstrating decreases in RANK is also small. Moreno et al. (37) have shown that the ability of IL-4 to irreversibly inhibit osteoclast formation in mouse bone marrow cells, human CD14+ monocytes, and RAW 264.7 cells is associated with decreased RANK expression. A decrease in RANK mRNA has also been noted after activation of gp130 by IL-6 (in combination with soluble IL-6 receptor) in mouse calvarial bones (38). Finally, decreases in RANK mRNA and protein expression have been observed in mouse calvariae, RAW 264.7 cells, and mouse spleen cells after treatment with IL-4 and IL-13 (39).

In the present investigation, a mouse calvarial bone culture model was used to assess the involvement of RANKL, OPG, and RANK expression in the stimulation of osteoclast differentiation and bone resorption by glucocorticoids. Because glucocorticoids have been shown to act in a synergistic fashion with D3 to stimulate bone resorption in mouse calvariae (16), RANKL, OPG, and RANK were also measured after costimulation of calvarial bones with dexamethasone and D3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and materials
Calvarial bones for the organ cultures were obtained from CsA mice from our inbred colony at Umeå University. The Institutional Animal Care and Ethics Committee approved all experimental studies. Mouse OPG fused to human IgG1 Fc (OPG/Fc chimera), recombinant mouse macrophage colony-stimulating factor (M-CSF), recombinant mouse RANKL, and the ELISA kits for RANKL and OPG protein analyses were purchased from R&D Systems (Abingdon, UK); essentially acid-free albumin, dexamethasone, hydrocortisone, and the kit for leukocyte acid phosphatase staining from Sigma Chemical Co. (St. Louis, MO); {alpha}MEM, fetal calf serum, TRIzol LS reagent, deoxyribonuclease I (amplification grade), and Superscript first-strand synthesis kit from Life Technologies Ltd. (Paisley, UK); [45Ca]CaCl2 and Thermo Sequenase-TM II DYEnamic ET terminator cycle sequencing kit from Amersham (Little Chalfont, Buckinghamshire, UK); oligonucleotide primers from Life Technologies or Applied Biosystems; HotStar Taq polymerase kit and QIAquick PCR purification kit from QIAGEN Ltd. (Crawley, West Sussex, UK); DNA free from Ambion Inc. (Austin, TX); first-strand cDNA synthesis kit and PCR core kit from Roche (Mannheim, Germany); fluorescent-labeled probes (reporter fluorescent dye VIC at the 5' end and quencher fluorescent dye TAMRA at the 3' end), TaqMan universal PCR master mix, and the kits for quantitative real-time PCR for vitamin D receptor (VDR) and glucocorticoid receptor from Applied Biosystems; culture dishes, multiwell plates, and glass chamber slides from Costar (Cambridge, MA) or Nunc International Corp. (Naperville, IL). RU 38486 [mifepristone; 17ß-hydroxy-11ß-(4-dimethylaminophenyl)17{alpha}-(1-propynyl)-estra-4, 9 dien-3-one] was a kind gift from Roussel-UCLAF (Romanville, France), indomethacin was obtained from Merck, Sharp & Dohme (Haarlem, The Netherlands), and D3 from Hoffmann-La Roche (Basel, Switzerland). D3 and indomethacin were dissolved in ethanol; the final concentration of ethanol never exceeded 0.1% and did not by itself affect 45Ca release in mouse calvariae. All other compounds were dissolved either in PBS or culture medium.

Bone resorption in vitro
Bone resorption was assessed by analyzing mineral mobilization in cultured mouse calvarial bones. Parietal bones from 6- to 7-d-old CsA mice were dissected and cut into four pieces. The bones were preincubated for 18–24 h in {alpha}MEM containing 0.1% albumin and 1 µmol/liter indomethacin. After preincubation, the bones were extensively washed and subsequently cultured for up to 96 h in multiwell culture dishes containing 1.0 ml of indomethacin-free medium, with or without test substances (40, 41). The bones were incubated in the presence of 5% CO2 in humidified air at 37 C. Mineral mobilization was assessed by analyzing the release of 45Ca from bones prelabeled in vivo as described previously (40, 41).

RNA isolation and first-strand cDNA synthesis
Half calvarial bones from 6- to 7-d-old mice were preincubated in {alpha}MEM/0.1% albumin containing 10–6 M indomethacin for 24 h and then washed extensively and subsequently incubated in {alpha}MEM/0.1% albumin with or without test substances for different time periods. Total RNA was extracted from half calvariae with TRIzol LS reagent by following the manufacturer’s protocol. The RNA was quantified spectrophotometrically, and the integrity of the RNA preparations was examined by agarose gel electrophoresis. Only RNA preparations showing intact species were used for subsequent analysis. Extracted total RNA was treated with deoxyribonuclease I to eliminate genomic DNA. One microgram of total RNA, after DNase treatment, was reverse transcribed into single-stranded cDNA with a first-strand cDNA synthesis kit using random primers. After incubation at 25 C for 10 min and 42 C for 60 min, the avian myeloma virus reverse transcriptase was denatured at 99 C for 5 min, followed by cooling to +4 C for 5 min. In some experiments, Superscript first-strand synthesis kit was used by following the manufacturer’s protocol with data obtained comparable with those in experiments in which a first-strand cDNA synthesis kit was used. The cDNA was kept at –20 C until used for PCR.

Semiquantitative PCRs
First-strand cDNA mixtures were amplified by PCR using a PCR core kit and PC-960G gradient thermal cycler (Corbett Research, Australia) or Mastercycler gradient (Eppendorf). The PCRs were performed using 1 µl template, 0.2 µM of each primer, 2.5 U Taq DNA polymerase, 1x PCR buffer, 0.2 mM deoxynucleotide triphosphates, and 1.5 mM MgCl2 (100 µl total volume), with the exception of those for carbonic anhydrase II and calcitonin receptor (CTR), which were performed with 1.25 mM MgCl2. The conditions for PCR of tartrate-resistant acid phosphatase (TRAP), CTR, carbonic anhydrase II, cathepsin K, and osteocalcin were: denaturing at 94 C for 2 min, annealing for 40 sec at 55 C (cathepsin K), 57 C (TRAP), 64 C (osteocalcin), or 67 C (carbonic anhydrase II, CTR), followed by elongation at 72 C for 90 sec; in subsequent cycles denaturing was performed at 94 C for 40 sec. The GenBank accession numbers, sequences of the primers, and positions for the 5' and 3' ends of the nucleotides for the predicted PCR products of TRAP, CTR, carbonic anhydrase, cathepsin K, and osteocalcin are given in Table 1Go. The estimated sizes of the PCR products were TRAP, 313 bp; CTR, 167 bp; carbonic anhydrase II, 410 bp; cathepsin K, 338 bp; osteocalcin, 198 bp. RT-PCR conditions, the sequences of the primers, GenBank accession numbers, numbers for the 5' and 3' ends of the nucleotides for the predicted PCR products, and the estimated sizes of the PCR products have been given previously for RANKL, RANK, OPG, and glyceraldehyde-phosphate dehydrogenase (36). Control assays included samples in which the reverse transcription reaction had been omitted and did not show any amplification (data not shown). PCR products were compared at the logarithmic phases of the PCRs. PCR products were electrophoretically size fractionated in 1.5% agarose gel and visualized using ethidium bromide. The identities of the PCR products were confirmed using a Thermo Sequenase-TM II DYEnamic ET terminator cycle sequencing kit with sequences analyzed on a 377 XL DNA sequencer (Applied Biosystems).


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  		TABLE 1. Sequences of primers and probes, GenBank accession numbers, and the numbers of the 5' and 3' ends of nucleotides for the predicted PCR products

 
Quantitative real-time PCRs
Quantitative real-time PCR analysis was performed using the TaqMan universal PCR master mix kit as described in detail previously (36). Reactions were performed in 20 µl with approximately 10–50 ng cDNA; 10 µl 2x universal PCR master mix; 100–150 nM of TaqMan probe; and 300–900 nM of primers. The GenBank accession numbers, sequences of the primers, and probes and positions for the 5' and 3' ends of the nucleotides for the predicted PCR products of CTR, TRAP, cathepsin K, RANKL, RANK, OPG, and ß-actin are given in Table 1Go. Amplification conditions used were an initial 2 min at 50 C, 10 min at 95 C, 40–50 cycles of denaturation at 95 C for 15 sec, and annealing/extension at 60 C for 1 min. For OPG an additional step of 57 C for 15 sec was added after denaturation. VDR and glucocorticoid receptor (GR) mRNA expressions were analyzed using commercially available kits designed by Applied Biosystems. The amplifications were performed using a 384-well thermal cycler. Control assays included samples in which the reverse transcription reaction had been omitted and did not show any amplification (data not shown). To control for variability in amplification due to differences in starting mRNA concentrations, ß-actin was used as an internal standard. The relative expression of target mRNA was computed from the target cycle threshold values and ß-actin cycle threshold values using the standard curve method (user bulletin #2, Applied Biosystems). The mRNA expression of ß-actin was not affected by either dexamethasone or D3.

RANKL and OPG protein analyses
The protein synthesis of RANKL and OPG was assessed by measuring the levels of RANKL and OPG in calvarial bones using commercially available ELISA kits. Calvarial bones were dissected from 6- to 7-d-old mice (CsA) and divided into two halves along the sagittal suture. After preincubation, a total of six to eight calvarial halves per group were individually incubated in 24-well plates in the absence or presence of dexamethasone (10–7 M) for 24 h in one set of experiments. In another set of experiments, the bone were incubated for 24 h in the absence and presence of dexamethasone (10–7 M), D3 (10–9 M), or D3 + dexamethasone. Periosteal and endosteal calvarial cells were lysed with 0.2% Triton X-100, and the extracted bone samples were analyzed using the manufacturer’s protocols for the ELISAs. The sensitivities of the immunoassays are 5 pg/ml.

Statistics
Statistical analysis was performed using the nonparametric Kruskal-Wallis/Mann-Whitney U test or Student’s t test with or without Bonferroni correction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of dexamethasone on the mRNA expression of osteoclast and osteoblast markers in mouse calvariae in vitro
Semiquantitative RT-PCR revealed that treatment of mouse calvarial bones for 48 h with dexamethasone (10–7 M) resulted in increased expression of mRNA for the osteoclast markers, TRAP, CTR, cathepsin K, and carbonic anhydrase II (Fig. 1AGo). Evaluation of osteocalcin revealed that the osteoblast marker was down-regulated (Fig. 1AGo).


Figure 1
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  		FIG. 1. Calvarial bones were cultured in the absence or presence of dexamethasone. Semiquantitative RT-PCR analyses of mRNA expression showed an increase of TRAP, CTR, cathepsin K, and carbonic anhydrase II and a decrease of osteocalcin after 48 h (A). Data were obtained by pooling cDNA from five bones, and RT-PCR analyses were performed twice on all the samples. Time-course studies of CTR mRNA expression measured with quantitative real-time PCR showed significant stimulation (*, P < 0.05; **, P < 0.01) by dexamethasone (10–7 M) at 3, 6, 24, and 48 h (B). Significant stimulation (**, P < 0.01) was also seen with increasing concentrations of dexamethasone (10–9 to 10–6 M; C). Additionally, TRAP (D) and cathepsin K (E) mRNA expression were significantly increased (*, P < 0.05; **, P < 0.01) in a concentration-dependent manner. The stimulatory effect of dexamethasone (10–7 M) on CTR mRNA was inhibited by addition of RU 38 486 (10–6 M; 24 h; F). Values in B–F represent the means for five to 10 individual calvarial bones, and SEM is shown as vertical bars when larger than the radius of the symbol. GAPDH, Glyceraldehyde-phosphate dehydrogenase.

 
Quantitative real-time PCR analysis demonstrated that dexamethasone (10–7 M) caused a time-dependent increase of CTR mRNA expression, with a statistically significant stimulation (P < 0.01) observed at 3–48 h (Fig. 1BGo). The stimulatory effect of dexamethasone on CTR mRNA at 24 h was dependent on the concentration of the glucocorticoid, with a statistically significant (P < 0.01) response obtained at 10–9 M (Fig. 1CGo). Similarly, expression of mRNA for TRAP (Fig. 1DGo) and cathepsin K (Fig. 1EGo) was enhanced in a concentration-dependent manner by dexamethasone.

The stimulatory effect of dexamethasone (10–7 M) on CTR mRNA at 24 h was abolished by the glucocorticoid receptor antagonist RU 38 486 (10–6 M; Fig. 1FGo).

Effects of OPG on glucocorticoid-induced release of 45Ca from mouse calvariae in vitro
The stimulatory effects of dexamethasone (10–6 M) and hydrocortisone (10–6 M) on 45Ca release from mouse calvarial bones observed after 96 h of stimulation were abolished by simultaneous treatment with OPG (300 ng/ml; Fig. 2Go).


Figure 2
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  		FIG. 2. Stimulation of percentage 45Ca release by 10–6 M hydrocortisone and 10–6 M dexamethasone in the absence and presence of OPG (300 ng/ml). Neonatal calvarial bones were preincubated for 24 h and then cultured in the absence and presence of test substances for 96 h. Values are based on means of six bones, and SEM is shown as vertical bars. Greater (*, P < 0.05) 45Ca release occurred in hydrocortisone and dexamethasone groups in comparison with untreated controls. The stimulatory effects of the two glucocorticoids were significantly (**, P < 0.01) inhibited by simultaneous treatment with OPG.

 
Effects of dexamethasone on RANKL, RANK, and OPG mRNA in mouse calvariae in vitro
Semiquantitative RT-PCR revealed that dexamethasone (10–7 M) treatment of mouse calvariae for 48 h resulted in increased mRNA expressions of RANKL, RANK and OPG (Fig. 3AGo). In agreement with previous studies (36, 38), D3 (10–9 M) increased mRNA expression of RANKL and RANK but decreased mRNA expression of OPG (Fig. 3AGo).


Figure 3
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  		FIG. 3. Semiquantitative RT-PCR analysis after 48 h of exposure of calvarial bones to dexamethasone (10–7 M) showed increased mRNA expression of RANKL, RANK, and OPG (A). D3 (10–9 M) increased expression of RANKL and RANK but decreased OPG expression (A). Data were obtained by pooling cDNA from five bones, and RT-PCR analyses were performed twice on the all samples. Quantitative real-time PCR showed significant (*, P < 0.05; **, P < 0.01) stimulations of mRNA for RANKL (B) and OPG (C) at 6, 24, and 48 h and RANK (D) at 24 and 48 h. Stimulation of RANKL, OPG, and RANK mRNA expression by dexamethasone at 24 h was dose dependent (10–9 to 10–6 M; E–G; *, P < 0.05; **, P < 0.01). Values in B–G represent the means for five to 10 individual calvarial bones, and SEM is shown as vertical bars when larger than the radius of the symbol. GAPDH, Glyceraldehyde-phosphate dehydrogenase.

 
Using quantitative real-time PCR, it was found that the stimulatory effects of dexamethasone (10–7 M) on RANKL, OPG, and RANK mRNA were time dependent. Significant stimulations of RANKL and OPG mRNA were observed at 6 h, whereas significant stimulation of RANK mRNA was observed at 24 h (Fig. 3Go, B–D). Stimulation of RANKL, OPG, and RANK mRNA expression was dependent on the concentration of dexamethasone (10–9 to 10–6 M), with increases in mRNA observed at and above 10–9 M (Fig. 3Go, E–G).

The stimulatory effects of dexamethasone (10–7 M) on RANKL, OPG, and RANK mRNA were inhibited by RU 38 486 (10–6 M; data not shown).

Effects of dexamethasone on RANKL and OPG protein in mouse calvariae in vitro
Stimulations of calvarial bones for 24 h with dexamethasone (10–7 M) resulted in a statistically significant (P < 0.001), 3-fold increase of RANKL protein in the bones (Fig. 4AGo). The protein levels of OPG in bones stimulated by dexamethasone for 24 h was 1.4-fold enhanced (Fig. 4BGo). However, the increased OPG protein level did not reach statistical significance but was in agreement with the increased OPG mRNA enhancement induced by dexamethasone (Figs. 3Go, C and F, and 5BGo).


Figure 4
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  		FIG. 4. Calvarial bones were treated with dexamethasone (10–7 M) for 24 h and the amount of RANKL (A) and OPG protein (B) analyzed using ELISA. Dexamethasone increased both RANKL and OPG protein, but only the effect on RANKL reached statistical significance (P < 0.001).

 

Figure 5
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  		FIG. 5. Calvarial bones were treated for 24 h with dexamethasone (10–7 M), D3 (10–9 M), or dexamethasone + D3. In one set of experiments, the mRNA expressions of RANKL, OPG, RANK, and CTR were assessed (A–C and E). In another set of experiments, the protein expression of RANKL was determined (D). Analysis with quantitative real-time PCR indicated that D3 significantly (**, P < 0.01) increased RANKL and RANK mRNA expression and significantly (**, P < 0.01) decreased OPG mRNA (A–C). Costimulation with dexamethasone and D3 increased RANKL mRNA expression synergistically (A). The decrease of OPG mRNA after D3 treatment was slightly but significantly (**, P < 0.01) reversed by the addition of dexamethasone (B). RANK mRNA expression was unaffected by cotreatment (C). RANKL protein was increased by both dexamethasone and D3, and cotreatment resulted in synergistic stimulation (D). Dexamethasone significantly stimulated CTR mRNA expression to a larger extent than D3 and cotreatment increased mean expression slightly more than additive (E). Values represent the means for nine to 11 individual calvarial bones, and SEM is shown as vertical bars.

 
Effects of dexamethasone, in the presence of D3, on RANKL, RANK, and OPG mRNA in mouse calvariae in vitro
In agreement with previous experiments (36, 38), treatment of mouse calvariae with D3 (10–9 M) for 24 h resulted in significant enhancements of RANKL and RANK mRNA expression and a significant decrease in OPG mRNA expression (Fig. 5Go, A–C). When calvariae were costimulated with dexamethasone (10–7 M) and D3 (10–9 M), the two hormones increased RANKL mRNA in a synergistic manner. In agreement with the data presented in Fig. 3Go, dexamethasone (10–7 M) enhanced OPG mRNA (Fig. 5BGo). When added to D3-stimulated bones, the inhibitory effect of D3 on OPG mRNA was slightly, but significantly (P < 0.01), reversed by dexamethasone (Fig. 5BGo). In contrast, when compared with the stimulatory effects of the hormones themselves, stimulation of RANK mRNA was unaffected by cotreatment with dexamethasone and D3 (Fig. 5CGo).

Cotreatment with dexamethasone (10–7 M) and D3 (10–9 M) caused a clear-cut synergistic stimulation of RANKL protein (Fig. 5DGo).

In comparison with D3 (10–9 M), a substantially larger stimulation of CTR mRNA was noted with dexamethasone (Fig. 5EGo). Cotreatment of calvariae with dexamethasone and D3 resulted in increased CTR mRNA (Fig. 5EGo).

Effects of dexamethasone, in the presence and absence of D3, on the mRNA expressions of vitamin D and glucocorticoid receptors
Treatment of calvariae for 24 h with dexamethasone (10–7 M) or D3 (10–9 M) resulted in 2.3- and 4.8-fold increase of VDR mRNA expression, respectively (Fig. 6AGo). Cotreatment with dexamethasone and D3 did not further enhance VDR mRNA.


Figure 6
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  		FIG. 6. Calvarial bones were treated for 24 h with dexamethasone (10–7 M), D3 (10–9 M), or their combination, and mRNA expression of vitamin D and glucocorticoid receptor was assessed using quantitative real-time PCR. Dexamethasone and D3 significantly enhanced VDR mRNA expression, but costimulation did not result in further enhanced expression (A). Dexamethasone, and costimulation with dexamethasone and D3 but not D3 alone, decreased GR mRNA expression (B).

 
The GR mRNA was significantly decreased by dexamethasone (10–7 M) but unaffected by D3 (10–9 M; Fig. 6BGo). Cotreatment also resulted in a significant inhibition of GR mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Increased concentrations of glucocorticoids can cause the development of Cushing’s syndrome, with severe osteoporosis (1, 2, 3, 4, 5, 6, 7, 8). In the past, the increase in bone resorption that occurred with excess glucocorticoids was suggested to be due to indirect actions of the compounds that led to the development of secondary hyperparathyroidism (10, 11, 12). Support for this idea came from experiments showing that addition of glucocorticoids to isolated rat osteoclast preparations caused inhibition of osteoclast activity and resorption, not stimulation (21). However, subsequent investigations with bone marrow cells (17), cocultures of stromal cells and osteoclast progenitor cells (18), fetal rat parietal bones (13), and neonatal mouse calvariae (14, 15, 16) established that glucocorticoids were capable of directly stimulating bone cells and increasing resorption, and it is now generally believed that the increase in resorption that occurs with glucocorticoid excess is due to direct actions of the steroids on bone.

In the current study, it was shown that stimulation of neonatal mouse calvarial bone resorption by dexamethasone is accompanied by time- and concentration-dependent increases in the expression of mRNA for markers of osteoclast differentiation: CTR, TRAP, cathepsin K, and carbonic anhydrase II. The enhancement of CTR mRNA by dexamethasone was inhibited by RU 38 486. RU 38 486 [mifepristone; 17ß-hydroxy-11ß-(4-dimethylaminophenyl) 17{alpha}-(1-propynyl)-estra-4, 9 dien-3-one] is a derivative of 19-nortestosterone that affects glucocorticoid binding to the receptor protein (42). Inhibition by RU 38 486 suggests that the stimulation of osteoclast mRNA by dexamethasone was mediated by glucocorticoid receptors and is in agreement with earlier observations showing that bone resorption stimulated by glucocorticoids in mouse calvariae can be inhibited by RU 38 486 (15). Additionally, it was found that mRNA expression of osteocalcin, an osteoblast marker, was decreased by dexamethasone. This suggests that the increase in calvarial bone resorption stimulated by dexamethasone was associated with decreased bone formation (9).

An interesting observation was that the stimulation of CTR mRNA expression by dexamethasone in calvariae was much greater than the increase caused by D3 (Fig. 5EGo). This observation is in agreement with previous findings in human and mouse osteoclasts (43, 44), in which it has also been demonstrated that the effect is due primarily to increased transcription of the CTR gene (43). This enhanced effectiveness of dexamethasone may help explain why glucocorticoids prevent the refractoriness noted with calcitonin treatment (45) and supports the finding that calcitonin inhibits resorption in mouse calvarial bones treated with hydrocortisone or dexamethasone without escape from inhibition being observed (15).

Stimulation of bone resorption can be due to enhanced osteoclast activity and/or increased osteoclast formation. The mouse calvarial bones used in the present study were preincubated for 24 h in medium containing indomethacin before being exposed to dexamethasone. Although osteoclasts are present in the calvariae when the bones are dissected, these cells are lost during the preculture period (46). Therefore, resorption in calvarial bones is dependent on formation of osteoclasts from the pool of mononucleated osteoclast progenitor cells present on both the periosteal and endosteal surfaces of the bone. Increased mRNA expression of CTR, TRAP, cathepsin K, and carbonic anhydrase is thought to be due to an effect of dexamethasone on osteoclast progenitor cell differentiation. GR{alpha} and GRß have been shown to be present in both stromal-like tumor cells and multinucleated osteoclast-like cells in human giant cell tumors (47). Thus, the effect of dexamethasone to stimulate osteoclast progenitor cell differentiation might be due to a direct effect of the glucocorticoid on these cells, or occur indirectly, via osteoblasts.

Potent stimulators of osteoclast formation and bone resorption such as PTH and D3 are believed to cause effects on bone-resorbing cells indirectly by enhancing RANKL expression and decreasing OPG expression in bone marrow stromal cells or in periosteal or endosteal osteoblasts (22, 23, 24). Evaluation of RANKL and OPG mRNA expression in mouse calvarial bones showed that stimulation of bone resorption by dexamethasone is associated with increased expression of RANKL. The enhancement of RANKL mRNA and protein by dexamethasone was similar to that noted with D3. This is the first observation of a glucocorticoid regulating RANKL in intact bone and is in agreement with the increase in RANKL that has been reported previously in a conditionally immortalized fetal human osteoblastic cell line and primary human osteoblast cultures after dexamethasone treatment (19, 28). In support of the importance of RANKL in the resorptive effect of dexamethasone, it was found that exogenous OPG blocked the increase in mouse calvarial bone resorption induced by the steroid.

In addition to RANKL, another factor that is thought to play an important role in bone resorption is M-CSF (22, 23, 24). M-CSF is produced in stromal cells/osteoblasts and increases colony expansion of monocyte/osteoclast progenitor cells (22, 23, 24). In an earlier study, Rubin et al. (48) found that dexamethasone can enhance expression of M-CSF in murine osteoblast-like cells.

Previous cell culture studies using human osteoblasts and human osteoblastic cell lines have demonstrated an inhibitory effect of glucocorticoids on OPG mRNA expression (19, 28, 29, 30). However, in the present study, dexamethasone increased expression of the decoy receptor in neonatal mouse calvarial bones. Stimulation of OPG by dexamethasone offers an explanation for why bone breakdown caused by dexamethasone in mouse calvariae is less than that caused by potent stimulators of resorption like PTH and D3, agents that decrease OPG expression. Dexamethasone is not the only example of a stimulator increasing OPG expression. Cytokines in the IL-6 family (IL-11, IL-6, leukemia inhibitory factor, and oncostatin M) increase both OPG and RANKL expression (36, 38). Like dexamethasone, these cytokines are also less effective stimulators of resorption when compared with PTH and D3. These findings emphasize the importance of comparing cellular studies with an experimental model in which bone resorption can be assessed.

Increased differentiation of osteoclast progenitor cells occurs after activation of RANK receptors (22, 23, 24). In the present study, it was found that treatment of calvarial bone with dexamethasone increased mRNA expression of RANK, suggesting that resorption stimulated by dexamethasone is dependent on enhanced RANK activation on osteoclast progenitor cells. Thus, the current data suggest that osteoclast differentiation and bone resorption in mouse calvariae treated with dexamethasone is characterized by increased expression of both RANKL and RANK, tempered by a concomitant increase in OPG expression. Moreover, increases in RANKL, OPG, and RANK expression noted after dexamethasone treatment of calvariae were all inhibited by RU 38 486, demonstrating the dependence of these responses on GRs.

Glucocorticoids have been shown previously to synergistically potentiate the bone resorptive effect of D3 in mouse calvariae (16) and to synergistically potentiate osteoclast formation in bone marrow cultures and cocultures of stromal cells and osteoclast progenitor cells (17, 18). Evaluation of D3 and dexamethasone cotreatment of calvariae revealed that RANKL mRNA and protein expression were synergistically increased. In addition, there was only a slight increase in OPG mRNA in comparison with that noted with D3 alone and no change in RANK mRNA from that noted with D3. These observations of RANKL, OPG, and RANK expression offer an explanation for why glucocorticoids and D3 can synergistically potentiate bone resorption.

Because enhanced regulation of VDR and GR expression might also play a role in the synergistic stimulation of bone resorption noted with dexamethasone and D3 cotreatment, we evaluated the mRNA expressions of the VDR and GR. VDR mRNA was enhanced by both dexamethasone and D3, with no synergism being observed. In contrast, GR was decreased by dexamethasone but not by D3. Thus, enhanced receptor expression does not appear to be associated with the synergistic stimulation of bone resorption found with dexamethasone and D3. The findings further suggest that the rather limited bone resorptive response to glucocorticoids may be partly explained by homologous down-regulation of GR.

In summary, experiments suggested that stimulation of mouse calvarial bone resorption by dexamethasone is due to both osteoblastic and osteoclastic actions of the glucocorticoid. In addition to increases in mRNA for osteoclast differentiation markers (CTR, TRAP, cathepsin K, and carbonic anhydrase II) increased RANKL and OPG mRNA and protein expression and enhanced RANK mRNA were found in cultured calvarial bones after dexamethasone treatment. An explanation for the synergistic potentiation of bone resorption that occurs when dexamethasone and D3 are combined was provided by experiments showing a synergistic potentiation of RANKL mRNA and protein expression and decreased OPG mRNA expression after cotreatment with the two agents.


   Acknowledgments
 
The skillful technical assistance of Mrs. Inger Lundgren, Mrs. Anita Lie, and the preparations of the figures by Mrs. Ingrid Boström are gratefully acknowledged.


   Footnotes
 
This work was supported by the Swedish Research Council, the Swedish Rheumatism Association, the Royal 80 Year Found of King Gustav V, SalusAnsvar, and the County Council of Västerbotten.

First Published Online April 13, 2006

Abbreviations: CTR, Calcitonin receptor; D3, 1,25(OH)2-vitamin D3; GR, glucocorticoid receptor; M-CSF, macrophage colony-stimulating factor; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-{kappa}B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; VDR, vitamin D receptor.

Received June 15, 2005.

Accepted for publication March 29, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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