SEARCH

SEARCH BY CITATION

Keywords:

  • Osteoprotegerin;
  • Strontium;
  • Bone Formation;
  • Bone Resorption;
  • Osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Strontium (Sr) exerts an anabolic and antiresorptive effect on bone, but the mechanism remains unknown. Osteoprotegerin (OPG) expressed by osteoblasts plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This study aims at evaluating the role of OPG in Sr-mediated inhibition of osteoclastogenesis and bone resorption. Six-week-old Opg knockout (KO) male mice and their wild-type (WT) littermates were treated orally with vehicle (Veh) or Sr compound (4 mmol/kg) daily for 8 weeks. Bone mass and microstructure in the lumbar spine (L4) and proximal tibia were analyzed with micro–computed tomography (µCT). Bone remodeling was evaluated with serum biochemical analysis and static and dynamic bone histomorphometry. Osteoclast differentiation potential and gene expression were analyzed in bone marrow cells. The findings demonstrate that Sr compound treatment results in greater bone volume and trabecular number than Veh treatment in WT mice. The anabolic response of trabecular bone to Sr treatment is attenuated in KO mice. Although Sr treatment significantly decreases in vitro osteoclastogenesis and bone resorption in WT mice, these effects are attenuated in KO mice. Furthermore, Sr treatment profoundly increases Opg gene expression in the tibias and OPG protein levels in the sera of WT mice. This study concludes that the inhibition of osteoclastogenesis and bone resorption is possibly associated with OPG upregulation by Sr treatment. © 2011 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Strontium (Sr) compounds (such as Sr ranelate) have been identified as new antiosteoporosis agents for their potential to decrease the vertebral and nonvertebral fracture risk in postmenopausal women.1–3 Unlike traditional antiosteoporosis agents, which either increase bone formation (by recombinant parathyroid hormones) or inhibit bone resorption (by bisphosphonates), Sr compounds have the potential to exert an uncoupling effect on bone formation and bone resorption. It has been shown that Sr ranelate treatment decrease the histomorphometric bone-resorption variables while maintaining or increasing the bone-formation variables in both animal studies4, 5 and clinical trials.1, 2, 6 However, the mechanism behind the dual effect of Sr on bone is not clear. Several in vitro studies have suggested that Sr has the potential to increase proliferation and differentiation of osteoblastic cells or osteoprogenitor cells.7–12 Moreover, one of our previous studies demonstrated that Sr increases the osteoblastic lineage differentiation of bone marrow stromal cells.13 In addition to the stimulatory effect on osteoblast proliferation and differentiation, it also has been shown that Sr decreases osteoclastic bone resorption.11, 14, 15 Several mechanisms for inhibited osteoclast activity have been proposed and explored, including inhibition of carbonic anhydrase II and vitronectin receptor expression, as observed in chicken bone marrow cells14; disruption of the osteoclast actin-containing sealing zone, as in murine spleen–derived osteoclasts11; and a decrease in ruffled border formation, as in murine bone marrow–derived osteoclasts.13 A more recent study has shown that Sr can induce apoptosis in mature osteoclasts of rabbits by stimulating the calcium-sensing receptors (CaSRs) at doses higher than 9 mM.16 Although these in vitro investigations have provided cellular and molecular evidence for the anabolic and antiresorptive effects of Sr on bone cells, they do not provide information on the cross-talk between osteoblasts and osteoclasts in response to Sr treatment.

Our recent in vivo study on ovariectomized rats showed that Sr treatment leads to a significant increase of Opg gene expression in long bones.17 Other in vitro studies suggested that Sr has the potential to modulate Opg expression in osteoblasts,18, 19 which also might contribute to the inhibitory effect of Sr on osteoclastogenesis. Based on the observation that osteoprotegerin (OPG) plays a pivotal role in controlling osteoclastogenesis and bone resorption by osteoblasts,20–25 we hypothesize that OPG may be involved in the cross-talk between osteoclasts and osteoblasts following Sr treatment. In this study, Opg gene–deficient mice were used to test this hypothesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Animal procedures

Six-week-old male Opg heterozygous mice with a C57BL/6J background were provided by the Shanghai Research Center for Biomodel Organisms. The heterozygous mice were crossed to obtain Opg knockout (KO) mice. Genotyping was performed by polymerase chain reaction (PCR) analysis based on previously described methods.26, 27 All the mice were housed under specific pathogen-free (SPF) conditions (22°C, 12 hour/12 hour light/dark, and 50% humidity) with free access to food pellets and tap water. After the mice were acclimated for 1 week, they were randomly assigned to the following four groups (n = 8/group): (1) WT mice treated with either vehicle (0.9% saline) supplemented with calcium (1.8 mmol/kg) (WT + Veh) or (2) Sr compound (Sr 4 mmol/kg and Ca 1.8 mmol/kg) (WT + Sr), (3) Opg KO mice treated with either vehicle supplemented with calcium (1.8 mmol/kg) (KO + Veh) or (4) Sr compound (Sr 4 mmol/kg and Ca 1.8 mmol/kg) (KO + Sr). The Sr compound used in this study was Sr-fortified tricalcium phosphate (Sr-TCP) powder, which was synthesized by partially substituting Ca in TCP with Sr. This Sr compound has been demonstrated to exert beneficial effects in a goat osteoporosis model by preventing trabecular bone deterioration.28, 29 The dosage adopted in this study has been demonstrated previously to exert anabolic effects on rat, mouse, and chicken bones.30–32 To examine whether the attenuation of the effects of Sr on Opg-deficient mice was independent of dosage, a follow-up study was performed using different doses of Sr compound (2 and 6 mmol/kg, n = 8/group). Both Veh and Sr compound were administrated orally daily in the mice and lasted for 8 weeks. Double fluorescence labeling with calcein (10 mg/kg sc) was performed on days 7 and 2 before euthanizing the mice. The lumbar spines (L4) and the tibias were harvested, and soft tissues were removed for further micro–computed tomographic (µCT) and bone histomorphometry analysis.

Biochemical analysis

The serum bone-formation and bone-resorption parameters were analyzed with biochemical assays. Serum samples were centrifuged at 3600 rpm for 5 minutes at 4°C and then stored at −80°C until use. Serum levels of amino-terminal propeptide of type 1 procollagen (P1NP), osteocalcin, C-terminal telopeptide of type 1 collagen (CTX-1), and tartrate-resistant acid phosphatase 5b (TRACP-5b) were measured by ELISA (IDS, Inc., Fountain Hills, AZ, USA). The intra- and interassay precision errors were less than 10% for all assays.

µCT measurement

Trabecular bone microarchitecture of the L4 and proximal tibia was analyzed with µCT (vivaCT40; Scanco Medical, Brüttisellen, Switzerland) at a 10.5-µm isotropic image resolution.33 The trabecular region was isolated from the cortical region in each 2D image by manual contouring analysis. Model-independent 3D measurement methods were used to calculate the following parameters: trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th*), trabecular number (Tb.N*), and trabecular separation (Tb.Sp*). For these structural parameters, an asterisk denotes that the analyses were done without an a priori assumption regarding the plate- or rodlike nature of trabecular bone structure.34

Bone histomorphometry

Both static and dynamic bone-formation and bone-resorption parameters were evaluated by histomorphometric analysis. After µCT scanning, both the L4 vertebrae and tibias were embedded in methyl methacrylate (MMA), and 4- and 8-µm sections were obtained. The 4-µm sections were prepared for TRACP staining to evaluate osteoclast number (N.Oc/B.Pm) and osteoclast surface (Oc.S/BS), and Goldner's trichrome staining was used to measure osteoid surface (OS/BS). The overall view of the trabecular bone volume of the L4 vertebra was performed with von Kossa/von Gieson staining on 8-µm-thick sections. The calculation of surface-referent bone-formation rate (BFR/BS) also was performed on 8-µm-thick sections under a fluorescence microscope. Histomorphometric analysis was performed using OsteoMeasure image-analysis software (OsteoMetrics, Atlanta, GA, USA). Bone histomorphometric variables were expressed according to the report of the American Society of Bone and Mineral Research Nomenclature Committee.35

Real-time PCR for gene expression

The osteoblast- and osteoclast-related genes in the bone and bone marrow were analyzed with real-time PCR. The osteoblast-related genes include runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), and osteopontin (Opn). The osteoclast-related genes include TRACP, cathepsin K, and osteoclast-associated receptor (Oscar). Long bones (tibias) and bone marrow were homogenized, and the total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA). The extracted RNA was dissolved in RNase-free distilled water. Then 2 µg of total RNA was first reverse transcribed into cDNA using the High Capacity cDNA Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Quantitative PCR for osteoblast and osteoclast gene expression was performed in 96-well plates, and the reaction volume was 20 µL/well, which included 2 × SYBR Green Master Mix (Applied Biosystems), diluted gene primers, and cDNA. The primer sequences were purchased from Invitrogen. Real-time PCR was run on ab ABI Prism 7300 (Applied Biosystems). Data were analyzed using the ΔΔCT relative quantification method, with each gene being normalized to GAPDH. The results were expressed as fold changes from the Veh-treated WT group.

Bone marrow mononuclear cells culture and bone-resorption pit assay

Bone marrow cells from the tibias and femurs were cultured for 24 hours in α modified essential medium (α-MEM) with 10% fetal bovine serum (FBS) in the presence of macrophage colony-stimulating factor (M-CSF; 100 ng/mL; PeproTech, Rocky Hill, NJ, USA). Nonadherent cells were collected and layered on a Ficoll-Hypaque gradient. Cells at the gradient interface were isolated and further cultured on 24-well dishes (1.0 × 104/well) in α-MEM with 10% FBS in the presence of M-CSF (50 ng/mL) and receptor activator of NF-κB ligand (RANKL; 100 ng/mL; PeproTech) for 6 days. Cells were fixed with 4% formaldehyde in PBS for 10 minutes. The fixed cells were washed with PBS three times and stained for TRACP according to the manufacturer's instructions (Sigma, St Louis, MO, USA). The number of TRACP+ multinucleated cells (three or more nuclei in each cell) was counted under the microscope.

To study the role of OPG in Sr-mediated inhibition of in vitro bone resorption, primary bone marrow mononuclear cells (2.0 × 103/well) were cultured in dentine slices (IDS, Ltd.) in a 96-well plate in the presence of M-CSF and RANKL. After 6 days of culture, cells were removed from the dentine slices by sonication for 5 minutes. The dentine slices were stained with 1% toluidine blue (Sigma) for 2 minutes, washed with distilled water, and observed under a light microscope. The resorption pit areas of dentine slices were quantified using an image-analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD, USA).

Opg and Rankl gene expression and serum protein level in WT and KO mice

The Opg and Rankl gene expression in bone and bone marrow from the proximal tibias in WT and KO mice after Sr treatment for 2 months was analyzed with real-time PCR assays. The serum soluble OPG and RANKL protein levels were analyzed with a mouse OPG or RANKL immunoassay (IDC Ltd.) according to the manufacturer's instructions.

Statistical analysis

Statistical analyses were performed using NCSS software (NCSS 2007; NCSS Statistical Software, Kaysville, UT, USA). All data were expressed as mean and SEM. The effects of genotype (WT versus KO), treatment (Sr versus Veh), and interaction were examined using two-way ANOVA for cross-sectional data. In the presence of a statistically significant effect of genotype, treatment, or genotype × treatment, post-hoc comparisons were performed, and Bonferroni corrections were made for all comparisons. A difference at p < .05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Effect of Sr treatment on bone microstructure in Opg WT and KO mice

The trabecular bone volume fraction (BV/TV) in the L4 vertebrae (Table 1) and proximal tibias (Fig. 1A, B) were significantly lower in KO mice than in WT mice (−42% and −56%, respectively). Accompanying the reduced bone mass, both trabecular thickness (Tb.Th*) and trabecular number (Tb.N*) in the L4 vertebrae (Table 1) and proximal tibias (Fig. 1C, E) were significantly lower in KO mice than in WT mice (−23% and −24% for the L4 and −8% and −17% for the proximal tibias, respectively). In WT mice, Sr compound treatment significantly increased BV/TV and Tb.N* in the L4 vertebrae (+20% and +18%; Table 1) and proximal tibias (+19% and +21%; Fig. 1B, E). In contrast, in KO mice, Sr compound had no significant effect on BV/TV or Tb.N* (Fig. 1B, E, Table 1). For the other bone microstructure parameters (ie, Tb.Th* and Tb.Sp*), Sr compound treatment did not have any significant effect in either WT or KO mice when compared with the vehicle treatment (Fig. 1C, D, Table 1).

Table 1. Trabecular Bone Microarchitecture in L4 Vertebrae in Opg WT and KO Mice After Sr Compound Treatment for 8 Weeks
GroupBV/TV (%)Tb.Th* (mm)Tb.Sp* (mm)Tb.N* (1/mm)
  1. BV = bone volume; TV = tissue volume; Tb.Th* = trabecular thickness; Tb.Sp* = trabecular separation; Tb.N* = trabecular number; NS = not significant.

WT + Veh27.3 ± 3.30.067 ± 0.0070.657 ± 0.0713.8 ± 0.4
WT + Sr32.7 ± 3.80.070 ± 0.0050.594 ± 0.0494.5 ± 0.5
KO + Veh15.8 ± 2.10.056 ± 0.0060.698 ± 0.0762.9 ± 0.3
KO + Sr17.3 ± 2.50.063 ± 0.0070.667 ± 0.0683.1 ± 0.2
Two-way ANOVA
 Genotype<.001<.001.02<.001
 Treatment.003.04NS.003
Sr vs.Veh
 WT.002NSNS.002
 KONSNSNSNS
thumbnail image

Figure 1. Trabecular bone microstructure in Opg WT and KO mice after Sr compound treatment for 8 weeks. (A) Representative 3D images of bone microstructure in the proximal tibias in Veh- or Sr-treated Opg WT and KO mice. Scale bar = 0.5 mm. Quantification of trabecular bone volume fraction (BV/TV) (B), trabecular thickness (Tb.Th*) (C), trabecular separation (Tb.Sp*) (D), and trabecular number (Tb.N*) (E) in the proximal tibias in WT or KO mice was determined from the µCT measurement.

Download figure to PowerPoint

Effect of Sr treatment on bone turnover markers in Opg WT and KO mice

Biochemical analysis demonstrated that serum bone-formation markers (ie, osteocalcin and P1NP) and bone-resorption markers (ie, TRACP-5b and CTX-1) were significantly higher in KO mice than in the WT controls (+18%, +80%, +75%, and +135%, respectively). In WT mice, serum osteocalcin level was significantly higher (+20%), whereas TRACP-5b and CTX-1 levels in the Sr compound group were significantly lower (−35% and −19%) than in the Veh group. In KO mice, serum CTX-1 level was found to be significantly decreased after Sr treatment (−25%), whereas serum osteocalcin (+36%), P1NP (+5%), and TRACP-5b (−10%) levels were not significantly different between the Sr and Veh groups (Table 2).

Table 2. Serum Bone-Formation Markers and Bone-Resorption Markers in Opg WT and KO Mice After Sr Compound Treatment for 8 Weeks
GroupOsteocalcin (ng/mL)P1NP (ng/mL)TRACP-5b (U/L)CTX-1 (U/L)
  1. P1NP = amino terminal propeptide of type 1 procollagen; CTX-1 = C-terminal telopeptide of type 1 collagen; TRACP = 5b = tartrate-resistant acid phosphatase 5b; NS = not significant.

WT + Veh25.3 ± 3.06.1 ± 1.24.8 ± 0.97.4 ± 1.5
WT + Sr30.4 ± 3.67.3 ± 1.33.1 ± 0.76.0 ± 1.1
KO + Veh29.9 ± 4.210.8 ± 2.18.4 ± 1.617.4 ± 4.4
KO + Sr35.2 ± 6.411.3 ± 2.57.5 ± 1.113.3 ± 3.4
Two-way ANOVA
 Genotype.006<.001<.001<.001
 Treatment.003NS.004.01
Sr vs.Veh
 WT.009NS.001.05
 KONSNSNS.02

Effect of Sr treatment on histomorphometric parameters in Opg WT and KO mice

The trabecular bone mass was viewed with von Kossa/von Gieson staining, and the bone-resorption parameters N.Oc/B.Pm and Oc.S/BS were analyzed in trabecular bone by means of TRACP staining (Fig. 2A, B). N.Oc/B.Pm and Oc.S/BS were significantly higher in KO mice than in WT controls at the L4 vertebrae (+38% and +56%, respectively; Fig. 2C, D) and the proximal tibias (+29% and +34%, respectively; Table 3). In WT mice, N.Oc/B.Pm and Oc.S/BS in the L4 vertebrae (Fig. 2C, D) were significantly lower in the Sr compound group than in the Veh group (−23% and −14%, respectively). In KO mice, Sr compound treatment had no effect on N.Oc/B.Pm at either the L4 vertebrae (Fig. 2C) or the proximal tibias (Table 3). However, Sr compound treatment significantly decreased Oc.S/BS by 8% in the L4 vertebrae (Fig. 2D) and by 17% in the proximal tibias in KO mice (Table 3), respectively.

thumbnail image

Figure 2. Trabecular bone resorption in Opg WT and KO mice after Sr compound treatment for 8 weeks. (A) Representative images of trabecular bone in the L4 vertebrae with von Kossa/von Gieson staining. Scale bar = 0.5 mm. (B) Representative images of TRACP staining in the L4 vertebrae, which was performed to evaluate the osteoclast activity and bone resorption in the Veh- or Sr-treated Opg WT and KO mice. Scale bar = 0.1 mm. Bone-resorption parameters N.Oc/B.Pm and Oc.S/BS were quantified by OsteoMeasure image-analysis software (C, D).

Download figure to PowerPoint

Table 3. Trabecular Bone Histomorphometric Parameters in the Proximal Tibias in Opg WT and KO Mice After Sr Compound Treatment for 8 Weeks
GroupN.Oc/B.Pm (1/mm)Oc.S/BS (%)OS/BS (%)BFR/BS (µm/d)
  1. OS = osteoid surface; BS = bone surface; N.Oc = osteoclast number; B.Pm = bone perimeter; BFR = bone-formation rate; Oc.S = osteoclast surface.

WT + Veh6.2 ± 0.56.8 ± 1.12.8 ± 0.30.32 ± 0.04
WT + Sr5.8 ± 0.65.3 ± 0.85.8 ± 0.60.38 ± 0.05
KO + Veh8.0 ± 0.810.6 ± 2.03.0 ± 0.30.44 ± 0.05
KO + Sr7.3 ± 0.58.8 ± 1.63.4 ± 0.50.46 ± 0.06
Two-way ANOVA
 Genotype<.001<.001.03<.001
 Treatment.01.004<.001.02
Sr vs.Veh
 WTNSNS<.001.03
 KONS.04NSNS

The static bone-formation parameter OS/BS was analyzed with Goldner's trichrome staining in trabecular bone (Fig. 3A). The dynamic bone-formation parameter BFR/BS was analyzed in trabecular bone based on the observation of double fluorescence labeling (Fig. 3B). BFR/BS in the L4 vertebrae (Fig. 3D) and the proximal tibias (Table 3) were significantly higher in the KO mice than in WT controls (+25% and +37%, respectively). In WT mice, OS/BS in both the L4 vertebrae (Fig. 3C) and the proximal tibias (Table 3) were significantly higher in the Sr compound group than in the Veh group (+189% and +105%, respectively). Similarly, BFR/BS in the L4 vertebrae (Fig. 3D) and the proximal tibias (Table 3) were significantly higher in the Sr compound group than in the Veh group (+33% and +20%, respectively). In KO mice, Sr compound marginally increased OS/BS in the L4 vertebrae (+15%; Fig. 3C) but not in the proximal tibias (Table 3). Moreover, there was no significant difference in BFR/BS in the L4 vertebrae (Fig. 3D) or the proximal tibias (Table 3) between the Sr and Veh groups in KO mice.

thumbnail image

Figure 3. Trabecular bone formation in Opg WT and KO mice after Sr compound treatment for 8 weeks. (A) Representative images of new bone formation with Goldner's trichrome staining in the L4 vertebrae. Scale bar = 0.1 mm. (B) Representative images of double calcein labeling in the L4 vertebrae by fluorescence microscopy. Scale bar = 0.2 mm. The bone-formation parameter osteoid surface (OS/BS) and bone-formation rate (BFR/BS) were quantified by OsteoMeasure image-analysis software (C, D).

Download figure to PowerPoint

Effect of different doses of Sr treatment on the bone microstructure and histomorphometric parameters in Opg WT and KO mice

To exclude the possibility that the observed attenuation of the anabolic effect of Sr in KO mice depended on the Sr compound dosage, we repeated the experiment with another two doses and evaluated the bone microstructure and the static and dynamic histomorphometric parameters in the proximal tibias. Sr compound at 2 mmol/kg was found to significantly increase BV/TV and Tb.N* (+17% and +14%) in the proximal tibias of WT mice; however such an anabolic effect was not observed in the Sr-treated KO mice. Sr compound at 6 mmol/kg significantly increased BV/TV (+25%), Tb.N* (+31%), and BFR/BS (+18%) and decreased Oc.S/BS (−57%) in WT mice. The anabolic effect of Sr compound on bone (BV/TV and BFR/BS) was found to be attenuated in the Sr-treated KO mice (Table 4).

Table 4. Trabecular Bone Microstructure and Histomorphometric Parameters in the Proximal Tibias of Opg WT and KO Mice After Different Doses of Sr Compound for 8 Weeks
GroupBV/TV (%)Tb.Th* (mm)Tb.Sp* (mm)Tb.N* (1/mm)Oc.S/BS (%)BFR/BS (µm/d)
  1. BV = bone volume; TV = tissue volume; Tb.Th* = trabecular thickness; Tb.Sp* = trabecular separation; Tb.N* = trabecular number; Oc.S = osteoclast surface; BFR = bone-formation rate; NS = not significant. Sr-2 = Sr at dosage of 2 mmol/kg; Sr-6 = Sr at dosage of 6 mmol/kg.

WT + Veh25.9 ± 3.40.062 ± 0.0070.666 ± 0.0713.6 ± 0.46.6 ± 0.90.33 ± 0.04
WT + Sr-230.3 ± 3.50.068 ± 0.0060.615 ± 0.0594.1 ± 0.55.3 ± 0.90.36 ± 0.05
WT + Sr-632.3 ± 4.20.070 ± 0.0080.636 ± 0.0624.7 ± 0.64.2 ± 0.70.39 ± 0.04
KO + Veh16.2 ± 2.10.058 ± 0.0060.743 ± 0.0732.8 ± 0.310.7 ± 2.00.40 ± 0.05
KO + Sr-218.3 ± 3.00.061 ± 0.0070.667 ± 0.0723.0 ± 0.39.2 ± 1.70.46 ± 0.06
KO + Sr-619.1  ± 2.60.064 ± 0.0070.656 ± 0.0693.4 ± 0.48.6 ± 1.90.43 ± 0.05
Two-way ANOVA
 Genotype<.001.006.02<.001<.001<.001
 Treatment<.001NS.02<.001<.001.04
Sr-2 vs.Veh
 WT.04NSNS<.001NSNS
 KONSNSNSNSNSNS
Sr-6 vs.Veh
 WT.001NSNS<.001.007.03
 KONSNSNS.03.03NS

Effect of Sr treatment on osteoblast and osteoclast maturation and function in WT and KO mice

The mRNA levels for Alp, Tracp, cathepsin K, and Oscar were significantly higher in KO mice than in WT controls (+196%, +159%, +99%, and 47%, respectively). In WT mice, the mRNA level of Runx2 was significantly higher in the Sr compound group than that in the Veh group (+119%). In contrast, the mRNA level of cathepsin K was significantly lower in the Sr compound group than in the Veh group (−60%). The mRNA levels of Alp and Opn were increased marginally and the mRNA level of Oscar was decreased marginally in the Sr compound group compared with the Veh group (p = .06 for all). In KO mice, Sr compound treatment did not exert any significant influence on the examined mRNA levels when compared with the Veh group (Fig. 4).

thumbnail image

Figure 4. Expression of genes related to osteoblast and osteoclast differentiation and maturation in Opg WT and KO mice after Sr compound treatment for 8 weeks. RNA was extracted from bone and bone marrow from the tibias, and real-time PCR was performed to evaluate expression of genes associated with osteoblast differentiation and maturation (ie, Runx2, Alp, and Opn) or osteoclast differentiation and maturation (ie, Tracp, cathepsin K, and Oscar). The gene expression was normalized to GAPDH, and the results were expressed as fold changes from the Veh-treated WT group.

Download figure to PowerPoint

To evaluate ex vivo osteoclastogenesis and bone resorption after Sr treatment in WT and KO mice, bone marrow mononuclear cells were cultured and treated with M-CSF and RANKL. After 6 days, osteoclastic cells were identified with TRACP staining (Fig. 5A). The bone resorption in dentine slices was evaluated with toluidine blue staining. The number of osteoclastic cells and the bone resorption pit area were found to be significantly higher in the KO mice than in WT controls (+28% and +57%, respectively). In WT mice, Sr compound treatment significantly decreased the number of osteoclastic cells and the bone-resorption pit area (−33% and −24%, respectively). However, no significant change was observed between Sr compound and Veh groups in KO mice (Fig. 5B, C).

thumbnail image

Figure 5. Osteoclastogenesis and bone-resorptive potential of bone marrow mononuclear cells in Opg WT and KO mice after Sr compound treatment for 8 weeks. (A) Representative images of TRACP staining to evaluate the osteoclastogenesis of bone marrow mononuclear cells. Scale bar = 0.1 mm. (B) The number of TRACP+ multinucleated cells (three or more nuclei each cell) was counted. The primary bone marrow mononuclear cells (2.0 × 103/well) were cultured in dentine slices in a 96-well plate in the presence of M-CSF and RANKL. After 6 days of culture, cells were removed from the dentine slices and then stained with 1% toluidine blue. The resorbed pit areas of dentine slices were quantified under a light microscope (C).

Download figure to PowerPoint

Effect of Sr treatment on Opg/Rankl gene expression and protein secretion in WT and KO mice

As anticipated, Opg gene expression in whole bone and protein secretion in the sera were nearly undetectable in KO mice. In WT mice, Opg gene expression and protein secretion were significantly higher in the Sr compound group than in the Veh group (+122% and +30%, respectively; Fig. 6A, C). Rankl gene expression and protein secretion were significantly higher in KO mice groups than in WT controls (+187% and +492%, respectively; Fig. 6B, D). There was no significant difference for Rankl mRNA level and protein secretion between the Sr compound group and the Veh group in WT mice (Fig. 6B, D). However, in KO mice, RANKL protein secretion was 13% lower in the Sr compound group than in the Veh group.

thumbnail image

Figure 6. Opg and Rankl gene expression in the bone and bone marrow and their protein levels in the sera of Opg WT and KO mice after Sr compound treatment for 8 weeks. (A, B) RNA was extracted from bone and bone marrow from the tibias, and real-time PCR was performed to evaluate the mRNA levels of Opg and Rankl. The gene expression was normalized to GAPDH, and the results were expressed as fold changes from the Veh-treated WT group. (C, D) The amount of soluble OPG and RANKL proteins in the sera of Veh- or Sr-treated WT and KO mice was measured with mouse OPG or RANKL immunoassays.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

As a new antiosteoporosis agent, Sr ralenate has been reported to exert beneficial effects on osteoporotic bones in postmenopausal women1, 2, 6 and in ovariectomy-induced rat osteoporosis models.4 At the cellular level, the beneficial effects are based on the dual effect of Sr on osteoblasts and osteoclasts, two key cell types that control the process of bone remodeling. The fact that osteoblasts are able to regulate osteoclastogenesis through the OPG/RANKL system hints that OPG/RANKL signaling may be involved in the Sr-mediated antiresorptive effect. In this study, we investigated this hypothesis using an Opg-deficient mouse model. Our results have demonstrated that Sr has the potential to increase bone formation while decreasing bone resorption, thereby increasing the overall bone volume in WT mice. Interestingly, such a dual effect of Sr on the trabecular bone of WT mice was attenuated in KO mice irrespective of the Sr compound dosage.

At week 15, the Opg KO mice showed severe osteopenia and increased bone resorption coupled with increased bone formation, as demonstrated by serum biochemical and histomorphometric analysis in Opg KO mice, consistent with a previous report.36 The upregulation of osteoblast-related genes, the downregulation of osteoclast-related genes, and the greater osteoclastogenesis from bone marrow mononuclear cells in KO mice indicate that the observed bone loss is due to the dominance of bone resorption over bone formation in KO mice. The biochemical and histomorphometric analyses demonstrate the dual effect of Sr on trabecular bone in WT mice. At the molecular level, the anabolic effect of Sr on trabecular bone is found to be associated with increased mRNA levels of Runx2, the transcription factor for preosteoblasts. In contrast, the antiresorptive effect of Sr on trabecular bone is found to be associated with decreased mRNA levels of cathepsin K, a specific marker for osteoclasts.37 In addition, the numbers of multinuclear osteoclastic cells and bone-resorption pit areas are reduced after Sr compound treatment in WT mice, whereas there is no significant change in KO mice, suggesting that the inhibition of osteoclastogenesis and bone resorption for bone marrow mononuclear cells by Sr treatment is attenuated by OPG deficiency. Taken together, these data suggest that the demonstrated anabolic and antiresorptive effects of Sr on bone metabolism of WT mice are attenuated in KO mice.

Previous studies have suggested that Sr has an inhibitory effect on the key osteoclast differentiation markers and resorptive capacity in murine and chicken osteoclasts.11, 14–15 However, such a direct effect on osteoclasts can only partially contribute to the antiresorptive effect of Sr on bone, considering the much lower concentration of Sr in the blood and bone marrow than in the local bone microenvironment. The uptake and distribution of Sr in the bone matrix after oral administration make it likely that osteoblasts, compared with osteoclasts, have more opportunity to come into contact with the Sr. Previous studies on monkeys have shown that Sr deposition in newly formed bone is much higher than in old bone at both trabecular and cortical sites.38, 39 Consequently, the incorporation of Sr into the bone surface could create a microenvironment conducive for direct interaction between Sr and osteoblasts. Within this context, while exerting an anabolic effect on bone, Sr also may confer an antiresorptive effect on osteoclasts through osteoblasts. Indeed, in this study we have observed a significantly higher mRNA level of Opg in the whole tibias of the Sr-treated group than in the Veh-treated group of WT mice. More important, the serum level of OPG was significantly higher in the Sr-treated mice than in the Veh-treated WT mice. Thus Sr treatment results in a marked increase in the ratio of OPG/RANKL, inhibiting the potential of osteoclastogenesis and bone resorption in WT mice. On the other hand, in KO mice, such an antiresorptive effect of Sr treatment is attenuated by the inability to stimulate the Opg gene and protein expression in osteoblasts.

Previous in vitro studies have shown that Sr has the potential to increase Opg mRNA levels and protein secretion in human osteoblasts and concurrently reduce Rankl mRNA expression and membrane-associated protein levels of RANKL.18, 19 It has been suggested that CaSR is the possible signaling pathway.19 It is quite likely that an alternative signaling pathways may exist because knockdown of CaSR by means of small interfering RNA (siRNA) can only partially abolish the stimulatory effect of Sr on Opg and Rankl gene expression and protein secretion.19 Indeed, we have found that mRNA levels of coreceptors of Wnt and lipoprotein receptor–related proteins (LRP) 5/6 and active dephosphorylated β-catenin protein levels in long bones increase significantly after Sr treatment in WT mice. In addition, the upregulation of mRNA levels of coreceptors of Wnt and the stabilization of β-catenin were not attenuated in KO mice after Sr treatment (data not shown). Our in vivo evidence indicates the possibility that the inhibition of osteoclastogenesis and bone resorption by Sr treatment is associated with activation of the Wnt/β-catenin signaling pathway. Interestingly, a study from transgenic mice in which β-catenin was constitutively stabilized in differentiated osteoblasts has showed that β-catenin, together with T-cell factor (TCF) proteins, can regulate osteoblast expression of OPG.40 Whether the upregulation of OPG in long bones after Sr treatment in WT mice is associated with this signaling pathway needs further study.

This is the first in vivo study to evaluate the role of OPG in Sr-mediated inhibition of bone resorption. However, this study is not without limitations. First, we initiated treatment on 6-week-old male mice only, which cannot provide information about age- and sex-related trabecular bone changes in response to Sr treatment in WT and KO mice. Second, 6-week-old young mice are still in the bone-development stage. Although we have observed greater bone volume and bone formation parameters in the Sr-treated group than in the control group in WT mice, we cannot ignore the fact that skeletal development in growing young mice may contribute in part to increased bone volume. Therefore, this may limit the extrapolation of this finding to the adult skeletal system in human beings. Third, the duration of treatment is relatively short, and the long-term effects of Sr treatment on trabecular bone microstructure and bone remodeling in Opg WT and KO mice need further investigation in future studies.

In conclusion, our study has demonstrated that Sr compound treatment increases bone formation, decreases bone resorption, and therefore increases trabecular bone mass and microstructure in Opg WT mice, which is associated with increased Opg gene expression and protein secretion in osteoblastic cells in response to Sr treatment. Interestingly, as hypothesized, the dual effect of Sr on bone metabolism is found to be attenuated in Opg-deficient mice. Therefore, in addition to its direct inhibitory effect on osteoclastogenesis, Sr may possess the potential to exert an indirect inhibition on osteoclastogenesis and bone resorption through regulation of OPG expression. This cross-talk between osteoclasts and osteoblasts may shed light on the mechanisms for the dual effect of Sr ralenate on osteoporotic bone in postmenopausal women.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

All the authors state that they have no conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We would like to thank Dr Dong Zheng for his careful animal handling in this study. This study was supported by the Innovation and Technology Fund (ITF; Project Ref. No. GHP/009/06) and Hong Kong Research Grants Council (RGC) Grant HKU7147/07.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References