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Endocrinology Vol. 146, No. 10 4349-4361
Copyright © 2005 by The Endocrine Society

Stimulation of Resorption in Cultured Mouse Calvarial Bones by Thiazolidinediones

A. M. Schwab1, S. Granholm1, E. Persson1, B. Wilkes, U. H. Lerner and H. H. Conaway

Department of Physiology and Biophysics (A.M.S., B.W., H.H.C.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Department of Oral Cell Biology (S.G., E.P., U.H.L.), Umeå University, SE-901 87 Umeå, Sweden

Address all correspondence and requests for reprints to: Howard H. Conaway, Ph.D., Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 505, Little Rock, Arkansas 72205. E-mail: conawayhowardh{at}uams.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dosage-dependent release of 45Ca was observed from prelabeled mouse calvarial bones after treatment with two thiazolidinediones, troglitazone and ciglitazone. Release of 45Ca by ciglitazone was decreased by the osteoclast inhibitors acetazolamide, calcitonin, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate, and IL-4, but not affected by the peroxisome proliferator-activated receptor {gamma} antagonist, GW 9662, the mitotic inhibitor, hydroxyurea, or indomethacin. Enhanced expression of receptor activator of nuclear factor-{kappa}B ligand (RANKL) mRNA and protein and decreased osteoprotegerin (OPG) mRNA and protein were noted after ciglitazone treatment of calvariae. Ciglitazone and RANKL each caused increased mRNA expression of osteoclast markers: calcitonin receptor, tartrate-resistant acid phosphatase, cathepsin K, matrix metalloproteinase-9, integrin ß3, and nuclear factor of activated T cells 2. OPG inhibited mRNA expression of RANKL stimulated by ciglitazone, mRNA expression of osteoclast markers stimulated by ciglitazone and RANKL, and 45Ca release stimulated by troglitazone and ciglitazone. Increased expression of IL-1{alpha} mRNA by ciglitazone was not linked to resorption stimulated by the thiazolidinedione. Ciglitazone did not increase adipogenic gene expression but enhanced osteocalcin mRNA in calvariae. In addition to exhibiting sensitivity to OPG, data indicate that stimulation of osteoclast differentiation and activity by thiazolidinediones may occur by a nonperoxisome proliferator-activated receptor {gamma}-dependent pathway that does not require cell proliferation, prostaglandins, or IL-1{alpha} but is characterized by an increased RANKL to OPG ratio.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PEROXISOME PROLIFERATOR-activated receptors (PPARs) are a family of ligand-regulated transcription factors that belong to the nuclear hormone receptor superfamily (1). There are three PPARs: {alpha}, {delta} (also called ß), and {gamma} (2, 3, 4, 5). The predominant PPAR{gamma} isoform is PPAR{gamma}1. A second isoform, PPAR{gamma}2, is expressed principally in adipose tissue (6). Natural PPAR{gamma} ligands include fatty acids and fatty acid derivatives (7), such as eicosapentaenoic acid (8), 9- and 13-hydroxyoctadecadienoic acid (9), and 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PG-J2) (10). Numerous synthetic ligands have also been described. These include drugs such as the insulin-sensitizing thiazolidinediones (e.g. ciglitazone, troglitazone, rosiglitazone) (10, 11) and nonsteroidal antiinflammatory compounds like indomethacin and ibuprofen (12).

Mesenchymal stem cells (stromal cells) are capable of developing into adipocytes, osteoblasts, myoblasts, fibroblasts, and chondroblasts (13, 14). PPAR{gamma} stimulates adipocyte differentiation, and it has been posited that the increase in adipose tissue and decrease in osteoblast differentiation associated with bone loss in conditions such as aging may be occurring reciprocally, because of increased PPAR{gamma} activity (15).

Osteoclasts arise from precursor cells of the monocyte/macrophage lineage (16). Stromal cells/osteoblasts mediate the formation of fully differentiated bone resorbing osteoclasts from progenitor cells (16). Key factors regulating osteoclastogenesis include macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-{kappa}B ligand (RANKL) (16, 17, 18, 19). M-CSF is a product of stromal cells/osteoblasts that enhances macrophage and osteoclast survival. RANKL is a transmembrane protein produced in stromal cells/osteoblasts. RANKL plays an essential role in osteoclast differentiation and function by binding to receptor activator of NF-{kappa}B (RANK) on osteoclast progenitor cells. Mice that do not express RANKL or RANK or are deficient in M-CSF do not have functional osteoclasts and develop osteopetrosis (20, 21, 22, 23). The interaction between RANKL and RANK can be blocked by osteoprotegerin (OPG), a soluble protein released from stromal cells/osteoblasts that acts as a decoy receptor for RANKL. Mice with a targeted deletion of opg develop multiple fractures and have decreased trabecular bone volume and numerous osteoclasts (24).

After exposure to RANKL and M-CSF, osteoclast formation and bone resorption in bone marrow coculture experiments (human mesenchymal stem cells and CD34+ hematopoietic stem cells) (25) and osteoclastogenesis in primary murine myeloid cells and Raw cells (26) have been reported to be blocked by both the natural PPAR{gamma} ligand 15d-PG-J2 and a thiazolidinedione, ciglitazone. Because only CD34+ stem cells expressed PPAR{gamma} and no supporting cells were present with the murine myeloid cells or RAW 264.7 cells, these studies have indicated that an inhibitory action of the PPAR{gamma} ligands is specific to the osteoclast lineage. Experiments in mouse whole-bone marrow cultures have also pointed to an inhibitory role for the PPAR{gamma} ligands (27). In mouse whole-bone marrow cultures, it has been found that thiazolidinedione drugs can decrease the number of tartrate-resistant acid phosphatase (TRAP+) multinucleated osteoclasts formed in response to 1,25-(OH)2 vitamin D3 (D3) and PTH, and dose dependently decrease basal as well as D3 and PTH-induced resorption on dentine slices (27).

However, in contrast to the studies showing inhibition of bone resorption, thiazolidinedione treatment of cocultures of murine bone marrow-derived BMS2 adipocytes and primary bone marrow cells has been found to have no effect on the formation of TRAP+ osteoclasts in the presence of D3 (28). Furthermore, an investigation testing the effectiveness of BRL49653 (rosiglitazone) in intact and ovariectomized rats has shown no inhibition of bone resorption in intact animals and a substantial increase in bone resorption in estrogen-deprived animals (29).

Not all actions of compounds known to be PPAR{gamma} ligands are mediated by PPAR{gamma} activation (30, 31, 32, 33, 34, 35, 36, 37, 38). In the case of the thiazolidinedione, ciglitazone, inhibition of cholesterol biosynthesis in cultured Chinese hamster ovary cells (35), stimulation of MAP kinase cascades in astrocytes and preadipocytes (36), decreased growth and increased apoptosis of renal interstitial fibroblasts (37), and decreased expression of inhibitor of DNA binding (Id2) in human aortic smooth muscle cells (38) have all been reported previously as PPAR{gamma}-independent mechanisms.

The effects of PPAR{gamma} ligands on bone resorption and expression of RANKL, OPG, and RANK have not been evaluated in calvarial bone explants. In the present investigation, the neonatal mouse calvarial model was used to: 1) test the effects of thiazolidinediones and 15d-PG-J2 on bone resorption, 2) determine whether resorptive effects are PPAR{gamma} dependent or independent, and 3) correlate changes in resorption with expression of regulators of osteoclastogenesis, such as RANKL, OPG, and RANK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Animal experiments were conducted in accordance with accepted standards of humane animal care and use as deemed appropriate by the Animal Care and Use Committees of the University of Arkansas Medical Sciences Campus (Little Rock) and by Umea University, Sweden.

Materials
Recombinant mouse RANKL, mouse OPG fused to human IgG1 Fc (OPG/Fc chimera), recombinant human IL-1{alpha}, recombinant human IL-1 receptor antagonist protein (IRAP), and immunoassay kits for mouse OPG and mouse RANKL were purchased from R&D Systems (Abingdon, UK); fetal bovine serum from ICN Pharmaceuticals, Inc. (Costa Mesa, CA); ciglitazone, pioglitazone, troglitazone, acetazolamide, hydroxyurea (HU), GW 9662 (Sigma no. M-6191), and acid-free fetal BSA from Sigma Chemical Co. (St. Louis, MO); {alpha}-MEM, TRIzol LS Reagent, and oligonucleotide primers from Invitrogen/Life Technologies (Paisley, UK); fluorescent-labeled probes (reporter fluorescent dye VIC at the 5'end and quencher fluorescent dye TAMRA at the 3'end) and TaqMan Universal PCR Master Mix from Applied Biosystems (Warrington, UK); the first strand cDNA synthesis Kit and PCR Core kit from Roche (Mannheim, Germany); Triton X-100, HotStar Taq polymerase, and QIAquick purification kit from Qiagen/VWR International (Stockholm, Sweden); Thermo Sequenase-TM II DYEnamic ET terminator cycle sequencing kit from Amersham Biosciences (Uppsala, Sweden); [45Ca]CaCl2 from Amersham Life Science (Little Chalfont, Buckinghamshire, UK); 15d-PG-J2 from Calbiochem (La Jolla, CA); synthetic bovine PTH 1-34 from Bachem (Bubendorf, Switzerland); and culture dishes and multiwell plates from Costar (Cambridge, MA). Salmon calcitonin (CT) was kindly supplied by Novartis (Basel, Switzerland); indomethacin by Merck, Sharp & Dohme (Haarlem, The Netherlands); D3 by Hoffmann-La Roche (Basel, Switzerland); and the bisphosphonate 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) from Henkel KGaA (Dusseldorf, Germany).

Bone organ culture
Bone resorption was assessed by analyzing mineral mobilization in cultured mouse calvarial bones. Parietal bones from 6- to 7-d-old CD-1 or 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 120 h in multiwell culture dishes containing 2.0 ml indomethacin-free medium, with or without test substances (39). The bones were incubated in the presence of 5% CO2 in humidified air at 37 C.

Measurements of mineral release
Mineral mobilization was assessed by analyzing the release of 45Ca from bones prelabeled in vivo. In most experiments, 2- to 3-d-old mice were injected with 1.5 µCi 45Ca and the amount of radioactivity in bone and culture media was analyzed by liquid scintillation at the end of the culture period. For the time course experiments, the mice were injected with 12.5 µCi 45Ca, and radioactivity was analyzed at different time points by withdrawal of small amounts of culture media. Release of isotope was expressed as the percentage release of the initial amount of isotope (calculated as the sum of radioactivity in medium and bone after culture) (39). In some experiments, the data were recalculated, and the results were expressed as percentage of control, which was set at 100%. This allowed for accumulation of data from several experiments.

RNA isolation and first strand cDNA synthesis
Before RNA isolation, five calvarial halfs per group were preincubated for 18–24 h in {alpha}-MEM containing 0.1% albumin and 1 µmol/liter indomethacin before subsequent incubation in 24-well plates in the absence or presence of ciglitazone and D3. Total RNA was extracted from individual bones 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. One microgram of total RNA was reverse transcribed into single-stranded cDNA with a 1st Strand cDNA Synthesis Kit using oligo(dT)15 primers. After incubation at 25 C for 10 min and at 42 C for 60 min, the avian myeloblastosis virus reverse transcriptase was denatured at 99 C for 5 min. The cDNA was kept at –20 C until used for PCR.

Semiquantitative RT-PCR
For semiquantitative RT-PCR analysis, RNA from four to five bones per each group were pooled. The PCR for CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}), cathepsin K, GAPDH, IL-6, IL-1{alpha}, IL-11, LIF, osteocalcin, oncostatin M (OSM), PPAR{gamma}1, PPAR{gamma}2, TNF-{alpha}, TRAP, RANKL, and Wnt10b were performed using PCR standard protocol. In the PCR for IL-1{alpha}, the final MgCl2 concentration was changed from 1.5–1 mM. The conditions for PCR were denaturing at 94 C for 2 min, annealing at various temperatures for 40 sed, followed by elongation at 72 C for 60 sec; in subsequent cycles, denaturing was performed at 94 C for 40 sec. The PCRs for CT receptor (CTR), NF of activated T cells 2 (NFAT2), integrin ß3, and matrix metalloproteinase-9 (MMP-9) were initiated with hot start at 94 C for 15 min, using HotStar Taq polymerase. Annealing temperatures were 54 C (OSM), 57 C (C/EBP{alpha}, cathepsin K, GAPDH, IL-11, integrin ß3, LIF, MMP-9, and Wnt10b), 58 C (TRAP), 60 C (IL-1{alpha}), 61 C (NFAT2 and TNF-{alpha}), 63 C (IL-6, PPAR{gamma}1, and PPAR{gamma}2), 64 C (osteocalcin), 65 C (RANKL), and 67 C (CTR). The PCR for RANKL was performed with a step-down technology in which the primer annealing temperature was decreased by 5 C every five cycles down to 45 C. The sequences of primers, positions of the 5' and 3' ends of the predicted PCR products, GenBank accession numbers, and estimated fragment lengths are listed in Table 1Go. The expressions of these factors were compared at the logarithmic phase of the PCR. No amplification was detected in samples where the RT reaction had been omitted (data not shown). The PCR products were separated by electrophoresis in 1.5% agarose gels and visualized using ethidium bromide. The identity of the PCR products was confirmed using a QIAquick purification kit and a Thermo Sequenase-TM II DYEnamic ET terminator cycle sequencing kit with sequences analyzed on an ABI377 XL DNA sequencer.


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  		TABLE 1. Sequences of primers used in semiquantitative RT-PCR, positions of the 5' and 3' ends of predicted nucleotides, GenBank accession numbers, and estimated fragment length

 
Quantitative real-time PCRs
Quantitative PCR analyses on cDNA from individual bones of RANKL, RANK, OPG, TRAP, cathepsin K, NFAT2, and ß-actin mRNA were performed using the TaqMan Universal PCR Master Mix kit and ABI PRISM 7900 HT Sequence Detections System and software (Applied Biosystems, Foster City, CA) as described previously (40). The sequences of the primers and probes, the GenBank accession numbers, and the positions of the 5'and 3'ends of the predicted nucleotides are given in Table 2Go. 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 threshold cycle values and ß-actin threshold cycle values using the standard curve method (User Bulletin #2, Applied Biosystems).


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  		TABLE 2. Sequences of primers and probes used in quantitative real-time PCR, positions of the 5' and 3' ends of predicted nucleotides and GenBank accession numbers

 
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 (41). After preincubation, eight calvarial halves per group were individually incubated in 24-well plates in the absence or presence of 10–5 M ciglitazone and 10–8 M D3 for 48 h. After treatment with 0.2% Triton X-100, extracted bone samples were analyzed using the manufacturer’s protocols for the ELISAs. The sensitivities of the immunoassays are 5 pg/ml. D3 was used as a positive control and, as expected, resulted in an increased protein level of RANKL and a decreased level of OPG in calvarial bones.

Statistical analysis
Statistical analysis of multiple treatment groups was performed with Tukey’s pair-wise comparison after one-way ANOVA of logarithmic transformed data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of neonatal mouse calvarial bone resorption by thiazolidinediones
PPAR{gamma} ligands (ciglitazone, troglitazone, pioglitazone, and 15d-PG-J2) were tested in neonatal mouse calvarial bone cultures to determine whether they could stimulate 45Ca release. The results of this study are shown in Fig. 1AGo. All agents were tested at concentrations ranging from 10–7 to 5 x 10–5 M. Calvarial bones were cultured for 120 h with the test substances. Pioglitazone and 15d-PG-J2 had no significant affect on 45Ca release at any concentration tested. In contrast, significant stimulations of 45Ca release were noted with troglitazone (at 10–6 and 5 x 10–6 M) and ciglitazone (at 5 x 10–6, 10–5, and 5 x 10–5 M). Release of 45Ca caused by troglitazone and ciglitazone was biphasic, with peak release by each agent decreased at higher concentrations. The decrease in release may have been due to toxicity of the thiazolidinediones because at 10–4 M ciglitazone, only a very small amount of RNA could be extracted from calvarial bones.



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  		FIG. 1. Concentration-dependent stimulation of 45Ca release from neonatal mouse calvarial bones (A). Different concentrations of 15d-PG-J2, pioglitazone, troglitazone, and ciglitazone were added at time zero, and bones were cultured for 120 h. Values are based on data obtained in three different experiments, with each point representing the mean of 14–15 bones. To accumulate data from different experiments, the release of 45Ca in controls was set at 100%. Vertical bars, SEM. The release of 45Ca was significantly different from control release in bones treated with 10–6 and 5 x 10–6 M troglitazone and 5 x 10–6, 10–5, and 5 x 10–5 M ciglitazone. Time course (B) of 45Carelease from neonatal mouse calvarial bones treated with ciglitazone (10–5 M) and two different concentrations of PTH (10–10 and 10–8 M). The values are based on data obtained in two separate experiments, with each point representing the mean of 14–15 bones. Vertical bars, SEM. The releases of 45Ca caused by 10–10 M PTH and 10–5 M ciglitazone were significantly (**, P < 0.01) enhanced at 72 and 120 h. The response caused by 10–8 M PTH was significantly increased at 24, 72, and 120 h (**, P < 0.01).

 
A time-course study showed that 10–5 M ciglitazone significantly enhanced (P < 0.01) 45Ca release at 72 and 120 h (Fig. 1BGo). Additionally, two different concentrations of PTH (10–10 and 10–8 M) were found to cause time-dependent stimulations of 45Ca release (Fig. 1BGo). The response caused by 10–10 M PTH was significantly enhanced (P < 0.01) at 72 and 120 h (Fig. 1BGo), whereas that caused by 10–8 M PTH was significantly increased (P < 0.01) at 24 (the first time measured), 72, and 120 h.

The stimulation of 45Ca release in calvarial bones by ciglitazone in the presence of different inhibitors, CT, AHPrBP, acetazolamide, indomethacin, HU, and IL-4
Effects on 45Ca release caused by addition of CT (10–9 M) and AHPrBP (10–4 M) to calvariae treated for 144 h with 10–8 M PTH and 10–5 M ciglitazone are shown in Fig. 2AGo. AHPrBP resulted in inhibition of resorption caused by ciglitazone and the positive control, PTH, throughout the experimental period. Inhibition of both ciglitazone and PTH was also observed with CT, but unlike the inhibition found with the bisphosphonate, escape from the inhibitory action of CT occurred in calvarial bones treated with ciglitazone and PTH.



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  		FIG. 2. Stimulation of percent 45Ca release at 24–144 h by 10–5 M ciglitazone and 10–8 M PTH in the absence and presence of 10–9 M CT and 10–4 M AHPrBP (A). Data are derived from one experiment and based on means of six to eight bones. Vertical bars, SEM. After preculture of bones in indomethacin as described in Materials and Methods, bones were preincubated for an additional 24 h in control medium or medium containing either PTH (10–8 M) or ciglitazone (10–5 M) to initiate bone resorption. After this prestimulation period, CT and AHPrBP were added, and small aliquots of media were removed at 24–144 h for evaluation of 45Ca release. Control release of 45Ca was significantly (P < 0.01) different from release in ciglitazone- and PTH-treated calvariae at 48–144 h. Significant inhibition (P < 0.01) of ciglitazone and PTH treatment was noted in the presence of AHPrBP at 48–144 h. Significant inhibition (P < 0.01) of ciglitazone and PTH treatment was also noted in the presence of CT at 48–144 h. However, although the magnitude of inhibition caused by AHPrBP increased from 48–144 h, inhibition was not as pronounced, with escape occurring in calvarial bones treated with CT for 48–144 h. Effects on 45Ca release caused by addition of 10–4 M acetazolamide (B), 10–6 M indomethacin (C), 10–3 M HU (D), and 10 ng/ml IL-4 (E) to neonatal mouse calvarial bones treated with 10–5 M ciglitazone, 10–8 M PTH, or 10–8 M D3 for 120 h. Values are based on data obtained in two experiments with each inhibitor, with each point representing the mean of 10–14 bones. To accumulate data from different experiments, the release of 45Ca in controls was set at 100%. Vertical bars, SEM. Acetazolamide (B) and IL-4 (E) significantly (P < 0.01) inhibited the increases (P < 0.01) in 45Ca release stimulated by ciglitazone, PTH, and D3, but inhibition was not noted when either indomethacin (C) or HU (D) were added to calvariae treated with ciglitazone, PTH, and D3.

 
Fig. 2Go (B–E) shows the effects on 45Ca release caused by additions of 10–4 M acetazolamide, 10–6 M indomethacin, 10–3 M HU and 10 ng/ml IL-4, respectively, to calvarial bones treated with 10–5 M ciglitazone, 10–8 M PTH or 10–8 M D3 for 120 h. Significant decreases (P < 0.01) in 45Ca release were observed when the osteoclast inhibitors acetazolamide and IL-4 were added to calvarial bones treated with either PTH or D3, but no changes in mineral release stimulated by PTH and D3 were noted in the presence of the inhibitor of prostaglandin biosynthesis, indomethacin, or the inhibitor of DNA synthesis, HU. Similar results were obtained in calvarial bones treated with 10–5 M ciglitazone. Acetazolamide and IL-4 proved to be potent (P < 0.01) inhibitors of resorption stimulated by ciglitazone, whereas indomethacin and HU had no effect on 45Ca release caused by the thiazolidinedione.

The mRNA expression of RANKL, OPG, and RANK and protein formation of RANKL and OPG in neonatal mouse calvariae treated with ciglitazone
In agreement with earlier studies (41), real-time quantitative PCR analysis revealed that treatment for 48 h with 10–8 M D3 increased expression (P < 0.01) of RANKL mRNA in neonatal mouse calvariae (Fig 3AGo). In addition, there was a significantly increased (P < 0.01) mRNA expression of RANKL after treatment of calvarial bones for 48 h with 10–5 M ciglitazone (Fig. 3AGo). However, although 10–8 M D3 also caused an increased mRNA expression (P < 0.01) of RANK in calvarial bones at 48 h, no significant change in RANK mRNA expression was observed in 10–5 M ciglitazone-treated calvariae (Fig. 3BGo). In contrast, when the mRNA expression of OPG was evaluated, significant decreases (P < 0.01) were noted after treatment with both 10–5 M ciglitazone and the positive control, 10–8 M D3 (Fig. 3CGo).



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  		FIG. 3. The mRNA expression of RANKL (A), RANK (B), and OPG (C) and ELISA analysis of protein formation of RANKL (D) and OPG (E) in normal (control) calvarial bones and mouse calvariae treated with 10–5 M ciglitazone and 10–8 M D3. For the mRNA expression of RANKL (A), RANK (B), and OPG (C), data are derived from means of four to five bones treated for 48 h. Total RNA was extracted from each bone and cDNA synthesized. Real-time, quantitative PCR analysis was performed using cDNA from individual bones. RANKL, RANK, and OPG levels were normalized to ß-actin. The effects of ciglitazone and D3 were calculated by setting the ratios obtained in control bones to 100%. Vertical bars, SEM. Ciglitazone had statistically significant (P < 0.01) effects on the mRNA expression of RANKL and OPG. D3 had statistically significant (P < 0.01) effects on the mRNA expression of RANKL, RANK, and OPG. For the ELISA analysis of RANKL (A) and OPG (B) protein, calvarial bones were cultured for 48 h in the absence and presence of 10–5 M ciglitazone and 10–8 M D3. After culture, calvarial bones were extracted with 0.2% Triton X-100 for 24 h at room temperature. Values represent the means of eight calvarial halves per group. Vertical bars, SEM. Ciglitazone and D3 increased (P < 0.01) RANKL protein formation and decreased (P < 0.01) OPG protein formation.

 
Earlier studies (41) have shown that treatment with 10–8 M D3 increases RANKL protein formation, whereas decreasing OPG protein formation in neonatal mouse calvariae. A good correlation between mRNA expression and protein synthesis was also noted with ciglitazone. Treatment of calvarial bones for 48 h with 10–5 M ciglitazone resulted in a significantly increased (P < 0.01) protein level of RANKL and a significantly decreased (P < 0.01) protein level for OPG (Fig. 3Go, D and E).

The mRNA expression of CTR, TRAP, cathepsin K, MMP-9, integrin ß3, and NFAT2 in neonatal mouse calvarial bones treated with ciglitazone or D3
Data showing that inhibitors of osteoclast formation such as IL-4 can block calvarial bone resorption stimulated by ciglitazone suggest that the resorption is dependent on osteoclast differentiation. This was supported by semiquantitative RT-PCR analysis, which revealed that treatment of mouse calvarial bones with either 10–5 M ciglitazone or 10–8 M D3 for 48 h would increase mRNA expression of the osteoclast markers integrin ß3, TRAP, cathepsin K, MMP-9, NFAT2, and CTR (Fig. 4AGo). In Fig. 4Go (B–D), real-time, quantitative PCR analysis showed that 10–5 M ciglitazone and 10–8 M D3 treatment of calvarial bones increased (P < 0.01) mRNA expression of TRAP (B), cathepsin K (C), and NFAT2 (D).



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  		FIG. 4. Semiquantitative RT-PCR analysis showing mRNA expression of integrin ß3, TRAP, cathepsin K, MMP-9, NFAT2, and CTR after treatment with 10–5 M ciglitazone and 10–8 M D3 for 48 h (A). Real-time, quantitative PCR analysis showing the effects of 10–5 M ciglitazone and 10–8 M D3 on mRNA expression of TRAP (B), cathepsin K (C), and NFAT2 (D) in calvarial bones treated for 48 h. Levels of the markers were normalized to ß-actin. The effects of ciglitazone and D3 were calculated by setting the ratios obtained in control bones to 100%. Vertical bars, SEM. Ciglitazone and D3 significantly enhanced (**, P < 0.01) the mRNA expression of TRAP, cathepsin K, and NFAT2.

 
Inhibitory effect of OPG on 45Ca release stimulated by ciglitazone, troglitazone, and RANKL and mRNA expression of osteoclast genes increased by ciglitazone and D3
The decoy receptor, OPG, is thought to decrease osteoclastogenesis by binding RANKL and decreasing activation of RANK. OPG has been shown previously (40, 41) to be a good inhibitor of 45Ca release stimulated by agents such as D3, IL-11, and RANKL in neonatal mouse calvariae. In the present study, addition of OPG to cultured calvarial bones for 96 h caused inhibition of resorption stimulated by 10–5 M ciglitazone. The inhibition was concentration dependent, with statistical significance (P < 0.05) observed at 100 and 300 ng/ml OPG (Fig. 5AGo). OPG (300 ng/ml) also caused significant (P < 0.01) inhibition of 45Ca release stimulated by 10–6 M troglitazone (Fig. 5BGo) and RANKL (200 ng/ml; Fig. 5CGo)



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  		FIG. 5. Dose response of the inhibitory effect of OPG on 45Ca release stimulated by ciglitazone in mouse calvarial bones (A). Different concentrations of OPG were incubated with 10–5 M ciglitazone for 120 h. Significant inhibition (**, P < 0.05) of 45Ca release was observed at 100 and 300 ng/ml OPG. Each point represents the mean of six bones. Vertical bars, SEM. OPG (300 ng/ml) inhibited (**, P < 0.01) 45Ca release stimulated by 10–6 M troglitazone (B) and 200 ng/ml RANKL (C) in neonatal mouse calvarial bones. OPG was incubated with troglitazone and RANKL for 96 h. Values are based on six bones per group. Vertical bars, SEM. Semiquantitative RT-PCR analysis of calvarial bones showing that 300 ng/ml OPG decreased mRNA expression of RANKL stimulated by 10–5 M ciglitazone (D) for 48 h and the osteoclast markers (integrin ß3, TRAP, cathepsin K, MMP-9, NFAT2, and CTR) stimulated by 10–5 M ciglitazone (D) and 200 ng/ml RANKL (E) for 48 h.

 
When the ability of OPG (300 ng/ml) to block gene expression stimulated by 10–5 M ciglitazone for 48 h was evaluated, decreased mRNA expression of integrin ß3, TRAP, cathepsin K, CTR, NFAT2, MMP-9, and RANKL was noted (Fig. 5DGo). Similar results were observed after stimulation of gene expression by RANKL (200 ng/ml) for 48 h. OPG (300 ng/ml) decreased mRNA expression of integrin ß3, TRAP, cathepsin K, CTR, NFAT2, and MMP-9 (Fig. 5EGo) stimulated by RANKL.

Effects of ciglitazone on cytokine expression
IL-1, TNF, and members of the IL-6 family of cytokines (IL-6, IL-11, LIF, and OSM) are good stimulators of bone resorption (40, 41, 42) with actions that can suppress PPAR{gamma} activity (43, 44). Therefore, experiments were performed to determine whether bone resorption stimulated by ciglitazone might be indirect, due to increased expression of cytokines.

No change in the mRNA expression of either IL-6 or IL-11 was observed in three independent experiments testing ciglitazone (Fig. 6AGo). Similarly, expression of mRNA for LIF and OSM was unaffected by ciglitazone treatment for 48 h (Fig. 6AGo). In contrast, the mRNA expression of TNF-{alpha} was decreased and that of IL-1{alpha} increased in three experiments (Fig. 6AGo).



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  		FIG. 6. Semiquantitative RT-PCR analysis of mRNA expression of IL-6, IL-11, LIF, OSM, TNF-{alpha}, and IL-1{alpha} in calvarial bones treated for 48 h with 10–5 M ciglitazone (A). Ciglitazone exposure had no effect on mRNA of IL-6 family cytokines (IL-6, IL-11, LIF, and OSM) but decreased mRNA of TNF-{alpha} and increased mRNA expression of IL-1{alpha} (A). The lack of an effect by IRAP, the IL-1 receptor antagonist, on 45Ca release (**, P < 0.01) stimulated by 10–5 M ciglitazone (B). After treatment with 100 ng/ml IRAP for 3 h, calvarial bones were cultured for an additional 96 h with 100ng/ml IRAP and 10–5 M ciglitazone. Values are based on six bones per group. Vertical bars, SEM.

 
To determine whether IL-1{alpha} might be responsible for resorption stimulated by ciglitazone, calvarial bones were preincubated with IRAP for 3 h before addition of either 10–5 M ciglitazone or IL-1{alpha} for 96 h (45). IRAP blocked 45Ca release stimulated by IL-1{alpha} (data not shown) but not release stimulated by ciglitazone (Fig. 6BGo).

Effects of ciglitazone on adipogenic and osteoblastic gene expression
PPAR{gamma} increases adipogenesis and may suppress osteoblastogenesis (15). In calvarial bones treated for 48 h with 10–5 M ciglitazone, decreased mRNA expression for both PPAR{gamma}1 and PPAR{gamma}2 was found (Fig. 7Go). This was in contrast to treatment with 10–8 M D3, which had no effect on the mRNA expression of PPAR{gamma}1, but increased that of PPAR{gamma}2 (Fig. 7Go). When looking at other genes involved in adipogenesis, it was found that C/EBP{alpha} (46) was not affected by either ciglitazone or D3 treatment but that mRNA expression of Wnt10b (47) was decreased by both agents (Fig. 7Go). Evaluation of osteocalcin, a gene involved in osteoblast differentiation (48), revealed that mRNA expression was increased by both ciglitazone and D3 (Fig. 7Go).



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  		FIG. 7. Semiquantitative RT-PCR analysis of the mRNA expression of adipogenic markers (PPAR{gamma}1, PPAR{gamma}2, Wnt10b, and C/EBP{alpha}) and the osteogenic marker, osteocalcin, after treatment for 48 h with 10–5 M ciglitazone and 10–8 M D3. Ciglitazone decreased mRNA expression of PPAR{gamma}1, PPAR{gamma}2, and Wnt10b, had no effect on C/EBP{alpha}, and increased expression of osteocalcin. D3 decreased mRNA expression of Wnt10b, had no effect on the mRNA expression of C/EBP{alpha} or PPAR{gamma}1, and increased expression of PPAR{gamma}2 and osteocalcin.

 
Inability of GW 9662 to inhibit bone resorption stimulated by ciglitazone
Extensive use has been made of GW 9662, an irreversible inhibitor of PPAR{gamma}, to determine whether responses of various ligands are PPAR{gamma} independent or dependent (25, 38, 49, 50). Figure 8Go shows that treatment of calvarial bones with 10–5 and 10–6 M GW 9662 had no effect on basal resorption, resorption stimulated by 10–5 and 3 x 10–6 M ciglitazone, or resorption stimulated by 10–8 M PTH for 96 h. These results suggest that bone resorption stimulated by ciglitazone occurs by a PPAR{gamma}-independent mechanism.



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  		FIG. 8. The inability of GW 9662, an irreversible inhibitor of PPAR{gamma}, to alter resorption in normal (control) calvarial bones and calvarial bones treated with either ciglitazone or PTH for 96 h. Data are derived from means of five to six bones The effects of ciglitazone and PTH were calculated by setting the ratios obtained in control bones to 100%. Vertical bars, SEM. GW 9662 (10–5 and 10–6 M) had no statistical effect on control resorption, resorption stimulated by 3 x 10–6 and 10–5 M ciglitazone, or resorption stimulated by 10–8 M PTH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Three thiazolidinediones (ciglitazone, troglitazone, and pioglitazone) and a natural PPAR{gamma} ligand, 15d-PG-J2, were evaluated for their ability to stimulate 45Ca release from prelabeled neonatal mouse calvarial bones. There were no changes in 45Ca release noted when either 15d-PG-J2 or pioglitazone was tested, but increases in 45Ca release were found after treatment with both ciglitazone and troglitazone. Release of 45Ca after treatment of calvarial bones with ciglitazone and troglitazone was biphasic, with peak release of each drug (5 x 10–6 M for troglitazone and 10–5 M for ciglitazone) depressed at increased concentrations. This decrease in release may have been due to toxicity of the compounds because it was found that only a very small amount of RNA could be extracted from calvarial bones treated with 10–4 M ciglitazone.

The greatest increase of 45Ca release in calvarial bones was observed with ciglitazone, the prototype thiazolidinedione (51). Resorption stimulated by ciglitazone was blocked by several well-documented inhibitors of osteoclastic bone resorption, the carbonic anhydrase inhibitor, acetazolamide (52), the bisphosphonate, AHPrBP (53), the polypeptide hormone, CT (54), and IL-4 (26, 55). Inhibition by these agents suggests that the resorptive action of ciglitazone in neonatal mouse calvarial bones was mediated by stimulation of osteoclast differentiation and activity.

Unlike the sustained inhibition of bone resorption found with acetazolamide, AHPrBP, and IL-4, the inhibition by CT was transient. This is characteristic of the escape phenomenon (56, 57) known to occur with CT.

Indomethacin had no effect on 45Ca release stimulated by ciglitazone, suggesting that the resorptive effect of ciglitazone was not dependent on prostaglandin biosynthesis, a view supported by the observation that ciglitazone did not enhance mRNA expression of cyclooxygenase-2 (data not shown). In addition to inhibiting prostaglandin production, indomethacin can serve as a PPAR{gamma} ligand (12). However, the concentration of indomethacin needed for PPAR{gamma} activation is significantly higher (58) than the concentration (10–6 M) employed to inhibit cyclooxygenase activity (40, 59).

A mitotic inhibitor, HU, was also found to have no effect on the release of 45Ca in bone explants treated with ciglitazone. The resorptive actions of agents such as the thyroid hormones (60) and transforming growth factor-ß (61) are inhibited by mitotic inhibitors in mouse calvariae. In contrast, osteolytic compounds like PTH and D3 can stimulate calvarial bone resorption by mechanisms that are not dependent on cell replication (62). Thus, ciglitazone may be stimulating resorption by enhancing differentiation and/or fusion of mononuclear preosteoclasts to terminally differentiated multinucleated osteoclasts at a postmitotic step in mouse calvarial bones.

To evaluate the possible involvement of cytokines in the resorptive action of ciglitazone, mRNA expression of IL-1{alpha}, TNF-{alpha}, IL-6, IL-11, LIF, and OSM was determined after ciglitazone treatment. TNF-{alpha} expression decreased, and no changes in mRNA expression of IL-6 cytokines (IL-6, IL-11, LIF, and OSM) were noted, but ciglitazone increased mRNA of IL-1{alpha}. However, 45Ca release stimulated by ciglitazone was not affected by an inhibitor (IRAP) of IL-1{alpha}, suggesting that IL-1{alpha} was not involved in the resorption stimulated by ciglitazone.

Constitutive expression of RANKL, OPG, and RANK mRNA was observed in the calvarial bones. No change in RANK mRNA expression was noted after ciglitazone treatment of calvariae, but the thiazolidinedione was found to increase RANKL and decrease OPG mRNA expression in calvarial explants. Furthermore, analyses revealed an increase in RANKL protein and a decrease in OPG protein after exposure to ciglitazone. The increased RANKL to OPG ratio observed after ciglitazone treatment provides an explanation for why the thiazolidinedione was found to be a good stimulator of calvarial bone resorption.

At the time of dissection, low numbers of osteoclasts were present in the 6- to 7-d-old calvariae used for study. These were lost during the preculture period when the calvarial bones are exposed to basic medium and indomethacin. During subsequent treatment with osteoclastic stimuli, enhanced 45Ca release is due to increased osteoclast formation from mononucleated osteoclast progenitor cells (Lerner, U. H., P. Lundberg, and M. Ransjö, unpublished data). In addition to stimulating expression of RANKL, ciglitazone increased expression of the osteoclast markers integrin ß3 (63, 64), TRAP (65, 66), cathepsin K (67), CTR (54), NFAT2 (68), and MMP-9 (69). RANKL also increased expression of integrin ß3, TRAP, cathepsin K, CTR, NFAT2, and MMP-9 in calvariae. Increased expression of the osteoclast markers by ciglitazone and RANKL is evidence that both agents stimulate osteoclast differentiation and function in calvariae and supports the role of RANKL in the stimulation of osteoclast differentiation and function by ciglitazone. Moreover, the importance of the RANKL-OPG-RANK system was further emphasized by observations showing that the stimulatory effects of troglitazone and ciglitazone on bone resorption, the stimulation of RANKL by ciglitazone, and the stimulation of osteoclast markers (integrin ß3, TRAP, cathepsin K, CTR, NFAT2 and MMP-9) by ciglitazone and RANKL were all inhibited by exogenous OPG.

In the murine marrow-derived mesenchymal progenitor cell line U-33/{gamma}2, treatment with either a thiazolidinedione, rosiglitazone, or natural ligands, 9-hydroxyoctadecadienoic acid and 15d-PG-J2, stimulates differentiation to adipocytes, while blocking differentiation to osteoblasts (15). To better characterize how ciglitazone stimulates resorption, mRNA expression of adipogenic and osteogenic markers was evaluated in calvariae after ciglitazone treatment. For the adipogenic markers, mRNA of Wnt10b (47), PPAR{gamma}1 and PPAR{gamma}2 were decreased, but there was no change in expression of C/EBP{alpha}, a stimulator of terminal adipocyte differentiation (46). Decreased expression of PPAR{gamma} may be sufficient to promote osteoblastogenesis (70) and the decrease in PPAR{gamma} mRNA and lack of an effect on C/EBP{alpha} noted with ciglitazone, coupled with stimulation of mRNA for osteocalcin, an important marker of osteoblast differentiation (48), support the contention that the thiazolidinedione increases osteoblastogenesis in calvarial bones.

There are numerous examples of non-PPAR{gamma}-dependent actions of natural and synthetic PPAR{gamma} ligands (30, 31, 32, 33, 34, 35, 36, 37, 38). In the present study, several lines of evidence suggest that the increases in resorption stimulated by troglitazone and ciglitazone in calvarial bones are not PPAR{gamma}-dependent effects. First, in dose-response experiments where troglitazone and ciglitazone were found to be stimulators of 45Ca release, the natural PPAR{gamma} ligand, 15d-PG-J2, and another thiazolidinedione, pioglitazone, had no effect on calvarial bone resorption. Second, stimulation of PPAR{gamma} can increase adipogenesis, but the effects of ciglitazone on mRNA of adipogenic and osteogenic markers in calvarial bones suggest that ciglitazone stimulates osteoblast differentiation. Third, selective activation of PPAR{gamma} is thought to occur when thiazolidinedione drugs stimulate PPAR receptors, but the irreversible PPAR{gamma} antagonist GW 9662 (40) had no effect on bone resorption stimulated by ciglitazone.

The reason why stimulation rather than inhibition of osteoclast-mediated resorption was observed with ciglitazone and troglitazone in the present study appears to be due to the ability of calvarial osteoblasts to produce a RANKL to OPG ratio after treatment that favors resorption, together with possible differences in periosteal osteoclast precursor cells and the less differentiated osteoclast precursor cells found in bone marrow and spleen cell cultures. It is possible that the lack of an inhibitory effect by either ciglitazone or troglitazone is due to differences in expression of PPAR{gamma}. Preliminary analysis has indicated that expression of PPAR{gamma}1 mRNA is similar in calvariae and mouse spleen cells after ciglitazone treatment but that expression of PPAR{gamma}2 mRNA is less in calvariae in comparison with the hematopoietic cells (Lerner, U. H., and H. H. Conaway, unpublished data).

Animal studies have suggested that bone loss can result from thiazolidinedione use. In agreement with our observation of ciglitazone and troglitazone stimulating bone resorption, an in vivo study in rats (29) has found no evidence for rosiglitazone to inhibit bone resorption in intact animals, whereas in ovariectomized rats, treatment with the thiazolidinedione caused significant stimulation of bone resorption, accompanied by decreased bone mass in the tibia, femur, and lumbar spine of the animals. Furthermore, although no effect on trabecular bone volume has been noted after troglitazone administration in mice (71), several studies have shown significant bone loss after treatment of mice with rosiglitazone (72, 73, 74, 75). Decreased bone formation has usually been suggested as the reason for the skeletal loss that occurs in mice treated with rosiglitazone, but a recent report has indicated that increased bone resorption can also play a prominent role (75). In humans, results have been mixed, with thiazolidinedione treatment being suggested to both protect against bone loss (76) and increase bone loss (77). Watanabe et al. (76) have reported that troglitazone treatment of type 2 diabetics for 1 yr decreases serum leptin and prevents bone loss. In contrast, results from the Health, Aging, and Body Composition cohort study (77) have suggested that thiazolidinedione (troglitazone, rosiglitazone, and pioglitazone) treatment of type 2 diabetic patients for longer than 24 months decreases bone mineral density of the femoral neck and total hip. It is possible that the differences noted after thiazolidinedione treatment in humans are related to the different durations of treatment, but additional studies will be necessary to clarify this point.


   Acknowledgments
 
The authors gratefully acknowledge the technical help of Mrs. Ingrid Bostrom, Inger Lundgren, and Birgit Andertun.


   Footnotes
 
This project was supported by grants from the Swedish Science Council (Project 07525), the Swedish Rheumatism Association, the Royal 80 Year Fund of King Gustav V, and the County Council of Vasterbotten and Salus Ansvar.

First Published Online June 30, 2005

1 A.M.S., S.G., and E.P. contributed equally to this study. Back

Abbreviations: AHPrBP, 3-Amino-1-hydroxypropylidene-1,1-bisphosphonate; C/EBP{alpha}, CCAAT/enhancer binding protein {alpha}; CT, calcitonin; CTR, CT receptor; D3, 1,25-(OH)2 vitamin D3; 15d-PG-J2, 15-deoxy-{Delta}12,14-prostaglandin J2; HU, hydroxyurea; IRAP, IL-1 receptor antagonist protein; M-CSF, macrophage colony-stimulating factor; MMP-9, matrix metalloproteinase-9; NF, nuclear factor; OPG, osteoprotegerin; OSM, oncostatin M; PPAR, peroxisome proliferator-activated receptor; RANK, receptor activator of NF-{kappa}B; RANKL, receptor activator of NF-{kappa}B ligand; TRAP, tartrate-resistant acid phosphatase.

Received May 18, 2005.

Accepted for publication June 21, 2005.


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