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Keywords:

  • OSTEOCLAST;
  • ACTIVE VITAMIN D;
  • ELDECALCITOL;
  • BONE METABOLISM;
  • RANKL

Abstract

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

Eldecalcitol (ED-71) is a new vitamin D3 derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED-71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED-71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED-71 (50 ng/kg body weight) to 8-week-old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED-71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle-arrested receptor activator of NF-κB (RANK)-positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED-71 affected neither the number of RANK-positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED-71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL-positive cell surface around the trabecular bone was significantly reduced in ED-71-treated mice than in the control mice. ED-71 administration also increased BMD in 12-week-old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED-71 increases BMD by suppressing RANKL expression in trabecular bone in vivo. © 2012 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
  10. Supporting Information

Bone remodeling is a dynamic process orchestrated by bone-forming osteoblasts and bone-resorbing osteoclasts. In normal bone remodeling, osteoclastic bone resorption is followed by osteoblastic bone formation through a coupling mechanism.1 Osteoporosis is a common skeletal disease involving a decrease in bone mineral density (BMD), bone quality, and bone strength. Osteoporosis is caused by an imbalance of bone resorption and bone formation, with the former exceeding the latter.2, 3

Osteoclasts are formed from hematopoietic precursors of the monocyte/macrophage lineage under the control of osteoblasts.4–6 Osteoblasts express two cytokines involved in osteoclastic differentiation; macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL).7–10 M-CSF is constitutively expressed by osteoblasts, whereas RANKL is expressed in osteoblasts in response to osteotropic hormones and cytokines such as 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3, calcitriol], parathyroid hormone (PTH), and prostaglandin E2.10, 11 Osteoblasts also produce osteoprotegerin (OPG), a soluble decoy receptor for RANKL, which inhibits osteoclastogenesis by blocking RANKL-RANK interaction.12–14 Osteoclast precursors express c-Fms (M-CSF receptor) and RANK (RANKL receptor), and differentiate into osteoclasts in response to M-CSF and RANKL.15, 16 We previously detected cell cycle-arrested quiescent osteoclast precursors (QOPs) as lineage-committed osteoclast precursors in vivo.17 QOPs detected in bone marrow as RANK-positive cells differentiated into osteoclasts in response to M-CSF and RANKL without cell cycle progression.

Vitamin D was originally discovered as an antirachitic agent capable of preventing a failure of bone mineralization. A deficiency of vitamin D results in rickets in the young and osteomalacia in adults. The administration of vitamin D to rachitic animals and humans cures impaired bone mineralization.18 It was therefore postulated that vitamin D directly stimulates osteoblastic bone formation and mineralization, but even now there is no direct evidence of this.

Vitamin D3 is first metabolized to 25-hydroxyvitamin D3 [25(OH)D3] in the liver, then to 1α,25(OH)2D3 in the kidney.18 In this pathway, renal 1α-hydroxylation is a rate-limiting step in the production of 1α,25(OH)2D3 and is strictly regulated by PTH, fibroblast growth factor 23 (FGF23), and the vitamin D status of animals.19–22 1α,25(OH)2D3 is now recognized as a steroid hormone that stimulates intestinal calcium transport activity through the nuclear vitamin D receptor (VDR).23

Yoshizawa and colleagues24 generated VDR knockout mice, and found no appreciable defects during development before weaning. After weaning, however, the VDR knockout mice failed to thrive, and hypocalcemia and infertility resulted. Both bone formation and mineralization were severely impaired as a typical feature of type II vitamin D-deficient rickets.25 Most of the animals died within 25 weeks after birth as a result of severe hypocalcemia. Unexpectedly, when the mice were fed a high-calcium diet, they developed normally even at week 50.26 Bone formation and mineralization in the VDR knockout mice fed a high-calcium diet were completely reestablished. From these results, it was concluded that the stimulating effect of 1α,25(OH)2D3 on bone formation and mineralization is rather indirect, occurring through stimulation of the intestinal absorption of calcium.

Although it appears paradoxical, 1α,25(OH)2D3 enhances bone resorption in vitro and in vivo by upregulating RANKL expression in osteoblast-lineage cells.10, 27–29 Nevertheless, 1α,25(OH)2D3 (calcitriol) and its prodrug, 1α-hydroxyvitamin D3 [1α(OH)D3, alfacalcidol], have been used as therapeutic agents for osteoporosis because they increase BMD and reduce the incidence of bone fracture.30 Interestingly, these beneficial effects are caused by the suppression of bone resorption. The mechanism by which calcitriol and alfacalcidol suppress osteoclastic bone resorption in vivo, however, remains unknown.

Eldecalcitol [ED-71, 1α,25-dihydroxy-2β-(3-hydroxypropoxy) vitamin D3] was discovered by in vivo screening of more than 900 analogs of 1α,25(OH)2D3 on the basis of BMD-promoting activity.31 Daily administration of ED-71 increased BMD and reduced bone turnover markers in normal, ovariectomized (OVX), and steroid-treated rats, and also in patients with osteoporosis.32–35 A phase III study in patients with osteoporosis showed that ED-71 increased BMD and reduced the incidence of bone fracture with a greater efficacy than alfacalcidol. On the basis of these clinical studies, ED-71 was approved for the treatment of osteoporosis in Japan in January 2011. ED-71 binds to VDR with less affinity [one-eighth of 1α,25(OH)2D3] but to serum vitamin D binding protein (DBP) with higher affinity (twofold) than 1α,25(OH)2D3.36, 37 The higher affinity of ED-71 for DBP may extend its half-life in serum. ED-71 suppresses PTH production only weakly compared with 1α,25(OH)2D3.38, 39 ED-71 is not converted to 1α,25(OH)2D3, but alfacalcidol is metabolized to 1α,25(OH)2D3 by liver mitochondrial 25-hydroxylase (CYP27A1).40 Such characteristics may explain the superiority of ED-71 over alfacalcidol.

The mechanism by which ED-71 inhibits bone resorption when administered daily to animals and humans is unclear. We have attempted to solve the discrepancy in the action of vitamin D compounds in vivo and in vitro. It is possible that ED-71 decreases the number of QOPs, which may suppress osteoclast formation and bone resorption. In the current study, we addressed this issue by evaluating the effects of ED-71 on osteoclastogenesis in vivo. We show here that the daily administration of ED-71 to mice increases BMD through a suppression of RANKL expression in osteoblasts and not through a decrease in the number of QOPs in bone. The discrepancy between in vitro and in vivo effects of ED-71 on osteoclastogenesis is discussed.

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
  10. Supporting Information

Reagents and antibodies

1α,25(OH)2D3 was purchased from Wako Pure Chemical Industries (Osaka, Japan). ED-71 was synthesized chemically at Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). RANKL (GST-RANKL) was generated in one of the authors' laboratories (Oriental Yeast). RANKL was labeled with fluorescein isothiocyanate (FITC) by using a fluorescein labeling kit (Dojindo Laboratories, Kumamoto, Japan) and used for fluorescence-activated cell sorting (FACS). A phycoerythrin (PE)-conjugated anti-F4/80 antibody, a PE-conjugated anti-CD115 (c-Fms) antibody, and an allophycocyanin (APC)-conjugated anti-CD11b antibody obtained from eBioscience (San Diego, CA, USA) were used for the FACS analysis. A biotin-conjugated anti-RANK antibody and a biotin-conjugated anti-RANKL antibody obtained from R&D Systems (Minneapolis, MN, USA) were used for the immunohistochemical analysis.

Animal experiments

Eight-week-old male C57BL6/J mice were purchased from Japan SLC (Shizuoka, Japan). The animals were given a normal rodent chow and tap water and acclimated to conditions for 1 week. They were then divided into four groups based on body weight. A pilot experiment showed that the daily administration of ED-71 at 50 and 100 ng/kg body weight to normal mice for 4 weeks significantly increased BMD in the femoral metaphysis. Therefore, the dosage of 50 ng/kg was employed for the in vivo experiment. ED-71 suspended in medium-chain triglyceride (Nisshin Oillio, Tokyo, Japan; 50 ng/kg) was administered daily by oral gavage for 2 and 4 weeks (n = 5). Medium-chain triglyceride was also administered as the vehicle to the control mice. Tetracycline and calcein were injected subcutaneously for bone labeling 5 and 2 days before sacrifice. Twelve-week-old OVX and sham-operated (Sham) mice of the C57BL6/J strain were obtained from Japan SLC. Five days after the operation, OVX mice were orally administered either vehicle (medium-chain triglyceride), ED-71 (50 ng/kg) or 1α,25(OH)2D3 (50 ng/kg), 5 days a week for 4 weeks (n = 6). Sham mice were also orally administered vehicle (n = 6). Blood and bone samples were collected at 6 hours after the last treatment. All experiments were carried out in accordance with the guidelines of the Matsumoto Dental University Experimental Animal Committee.

Biochemical analysis

Calcium concentrations and alkaline phosphatase (ALP) activity in serum were determined using commercially available assay kits (Calcium E-test Wako and Lab Assay ALP, Wako, Tokyo, Japan). Serum concentrations of tartrate-resistant acid phosphatase 5b (TRAP5b) were measured by using a mouse TRAP assay kit (Immunodiagnostic Systems, Boldon, UK).

Measurement of bone mineral density

Left femora were collected and fixed in 70% ethanol. Three-dimensional (3D) reconstructions of distal femora were obtained by micro-computed tomography (µCT) (µCT-40, Scanco Medical, Zurich, Switzerland). Tomographic measurements of BMD were performed by pQCT (XCT Research SA + ; Stratec Medizintechnik, Pforzheim, Germany). The bone was placed horizontally inside a tube and scanned using a voxel size of 0.07 mm. The scan line was adjusted using the scout view. The image analysis was carried out using integrated XCT 2000 software. A slice of the distal femoral metaphysis starting at 1 mm (male mice) or 0.6 mm (female mice) from the growth plate was scanned for the measurement of trabecular and cortical BMD.

Bone histomorphometry

Bone histomorphometry was performed with trabecular bone at the femoral metaphysis at the Ito Bone Histomorphometry Institute (Niigata, Japan). Tissue volume (TV, µm2), bone volume (BV, µm2), bone surface (BS, µm), single-labeled surface, double-labeled surface, interlabeling thickness, trabecular thickness (Tb.Th, µm), and osteoclast number (N.Oc, cells) were measured. These primary parameters were used to calculate bone volume (BV/TV, %), osteoclast surface (Oc.S/BS, %), osteoclast number (N.Oc/BS, cells/mm), osteoblast surface (Ob.S/BS, %), the mineral apposition rate (MAR, µm/day), and the bone formation rate (BFR/BS, mm3/mm2/year).

Flow cytometric analysis

Bone marrow cells obtained from mice were layered onto a lymphocyte-M gradient (Cedarlane Laboratories, Ontario, Canada). After centrifugation, mononuclear cells were collected and incubated with FITC-labeled RANKL, PE-conjugated anti-F4/80, PE-conjugated anti-CD115 (c-Fms), or APC-conjugated-anti-CD11b antibody for 30 minutes. Cells were washed two times and analyzed using a Cytomics FC500 (Beckman Coulter, Fullerton, CA, USA).

QOP in vitro assay

Bone marrow cells obtained from tibias were cultured in α-minimal essential medium (αMEM, Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; JRH Bio-sciences, Lenexa, KS, USA), 104 units/mL M-CSF (Kyowa Hakko-Kirin, Tokyo, Japan), and 5 nmol/L RANKL (GST-RANKL, Oriental Yeast, Tokyo, Japan) in the presence of 50 µmol/L hydroxyurea (MP Biomedicals LLC, Eschwege, Germany) for 4 days. Cells were fixed in 10% paraformaldehyde and stained for TRAP. TRAP-positive cells containing more than three nuclei were counted as osteoclasts.

Quantitative RT-PCR

The right femora of mice (n = 3) were excised, immediately soaked in TRIzol (Invitrogen, Carlsbad, CA, USA), and crushed with TissueLyser II (Qiagen, Hilden, Germany). Total RNA was extracted with a Purelink RNA mini kit (Ambion, Austin, TX, USA). The cDNA was synthesized from 285 ng of total RNA by a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). The reaction was performed at 42°C for 1 hour. The expression of mRNA in bone was detected using a Fast SYBR Green Master Mix protocol (Applied Biosystems LLC, Carlsbad, CA, USA). Target cDNA was amplified by 40 cycles (1 cycle: 95°C for 3 seconds, 60°C for 30 seconds) of PCR in the Applied Biosystems StepOnePlus. PCR primers for GAPDH, RANKL, OPG, and M-CSF of mice were obtained from Takara Bio (Shiga, Japan). GAPDH was used as a control. Data are represented relative to the control.

Immunohistochemical analysis

Paraffin-embedded samples (4 µm thick) of tibia were subjected to staining for TRAP and counterstaining with hematoxylin. For immunofluorescent staining, tibias were frozen in hexane using a cooling apparatus (PSL-1800, Tokyo Rikakikai, Tokyo, Japan) and embedded in a 5% carboxymethyl cellulose gel. The 5-µm-thick sections of undecalcified tibias were prepared using Kawamoto's film method (Cryofilm transfer kit, Finetec, Tokyo, Japan) and fixed in ice-cold 5% acetic acid in ethanol.41 The sections were stained with a biotin-conjugated anti-RANK antibody or a biotin-conjugated anti-RANKL antibody. Biotinylated antibodies were visualized with a Tyramide Signal Amplification kit for FITC (PerkinElmer, Norwalk, CT, USA). The sections were also stained for 4,6-diamidino-2-phenylindole (DAPI). The RANKL-positive cell surface (RANKLS, µm), RANK-positive surface (RANKS, µm), and bone surface (BS, µm) were measured using software (TRI/3D Bon, Ratoc, Tokyo, Japan).

Statistical analysis

Data represent the mean ± standard error of the mean (SEM). The statistical analysis was performed using Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA, USA). Statistical significance was determined with Student's t tests.

Results

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

ED-71 (50 ng/kg) was administered orally to normal mice everyday for 2 and 4 weeks. There was no significant difference in body weight (Fig. 1A) or physical activity between the ED-71-treated and the vehicle-treated mice at 2 and 4 weeks. The serum concentration of calcium was 9.51 ± 0.14 mg/dL and 9.71 ± 0.15 mg/dL in mice treated with ED-71 for 2 and 4 weeks, respectively (Fig. 1A). The concentration was significantly higher in the ED-71-treated mice than the vehicle-treated mice but was still within the normal range (9.0 to 10.0 mg/dL). These results indicate that the dosage of ED-71 employed in this study does not induce hypercalcemia.

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Figure 1. Effects of daily administration of ED-71 on body weight, serum calcium concentrations, and femoral bone structure in mice. ED-71 (50 ng/kg body weight) or vehicle was administered daily to 9-week-old male mice for 2 and 4 weeks. Blood and bone samples were recovered for further analyses. (A) Body weight and serum calcium concentrations were determined in mice treated with ED-71 or vehicle. (B) Three-dimensional µCT images of femurs were reconstructed in mice treated with ED-71 or vehicle for 4 weeks. (C) BMD of trabecular and cortical bones, bone volume/total tissue volume (BV/TV), and trabecular thickness (Tb.Th) in the distal femurs were measured in mice treated with ED-71 or vehicle. Values represent the mean ± SEM (n = 5 for each treatment group; *p < 0.05 versus vehicle group by Student's t test).

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Femurs were removed at 2 and 4 weeks and subjected to a bone structure analysis. Three-dimensional images of the trabecular-rich region of distal femurs in mice treated for 4 weeks were reconstructed using a µCT technique (Fig. 1B). Daily administration of ED-71 for 4 weeks increased trabecular bone volume compared with that of the vehicle-treated mice. Outcomes of pQCT (Fig. 1C, upper panels) confirmed that ED-71 administration increased BMD in the trabecular bone (upper left). However, there was no significant difference in cortical BMD between the ED-71-treated and the vehicle-treated mice (upper right). Bone histomorphometric analysis of the distal femoral metaphysis revealed that BV/TV and Tb.Th in mice treated with ED-71 for 4 weeks were significantly increased compared with the vehicle-treated mice (Fig. 1C, lower panels). Thus, the treatment of normal mice with ED-71 increased BMD in the trabecular bone but not in the cortical bone.

Effects of ED-71 administration on bone resorption were assessed by using TRAP staining and histomorphometric evaluation (Fig. 2). TRAP-positive cells were observed in the epiphyseal growth plate region and along the cancellous bone surface in mice treated with vehicle or ED-71 for 4 weeks (Fig. 2A). However, there were fewer TRAP-positive cells in the trabecular bone area in the ED-71-treated mice. Both Oc.S/BS and N.Oc/BS were significantly decreased in the ED-71-treated mice compared with the vehicle-treated mice (Fig. 2B). Serum levels of TRAP5b, a bone resorption marker, were also decreased significantly in the ED-71-treated mice (Fig. 2C). Using immunohistochemical staining, we have shown that RANK-positive cells can be detected along the bone surface as QOPs and mature osteoclasts. Immunologically stained RANK-positive cells were observed along the bone surfaces in ED-71- and vehicle-treated mice (Fig. 2D). When RANK-positive cell surface (RANKS) and bone surface (BS) were measured, RANKS/BS (µm/µm) was significantly decreased in ED-71-treated mice compared with the vehicle-treated mice (Fig. 2D).

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Figure 2. Effects of ED-71 administration on bone resorption in mice. ED-71 (50 ng/kg body weight) or vehicle was administered daily to 9-week-old male mice for 2 and 4 weeks. (A) Tibial sections prepared from mice treated with ED-71 or vehicle for 4 weeks were stained for TRAP. (B) Osteoclast surface/bone surface (Oc.S/BS) and osteoclast number/bone surface (N.Oc/BS) in distal trabecular bones of femurs were measured in mice treated with ED-71 or vehicle. (C) Serum concentrations of TRAP5b were measured in mice treated with ED-71 or vehicle. (D) Tibial sections prepared from mice treated with ED-71 or vehicle for 4 weeks were stained with an anti-RANK antibody (green) and DAPI (nuclei, blue). RANK-positive cell surface (RANKS)/bone surface (BS) was measured on trabecular bone. Values represent the mean ± SEM (n = 5 for each treatment group; *p < 0.05 versus vehicle group by Student's t test).

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The effect of ED-71 on bone formation was assessed by using bone histomorphometry based on the incorporation of double fluorochrome-labeled markers (Fig. 3). The width of the double fluorescent lines was reduced by the ED-71 treatment for 4 weeks (Fig. 3A). Both BFR/BS and Ob.S/BS in the distal femoral metaphysis were significantly lower in mice treated with ED-71 for 2 and 4 weeks than in the vehicle-treated mice (Fig. 3B). Serum ALP activity, a bone formation marker, was also lower in the ED-71-treated mice (Fig. 3C). Thus, the daily administration of ED-71 inhibited both bone resorption and bone formation. These results suggest that the increase in BMD induced by ED-71 is mainly because of the suppression of bone resorption.

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Figure 3. Effects of ED-71 administration on bone formation in mice. ED-71 (50 ng/kg body weight) or vehicle was administered daily to 9-week-old male mice for 2 and 4 weeks. (A) Tetracycline and calcein were injected subcutaneously into mice treated with ED-71 or vehicle for 4 weeks for bone labeling 5 and 2 days before sacrifice. Double labeling with tetracycline and calcein was assessed in femoral bone sections. (B) Mineral apposition rate (MAR), bone formation rate/bone surface (BFR/BS), and osteoblast surface/bone surface (Ob.S/BS) in distal trabecular bones of femurs were measured in mice treated with ED-71 or vehicle. (C) Serum ALP activity was measured in mice treated with ED-71 or vehicle. Values represent the mean ± SEM (n = 5 for each treatment group; *p < 0.05 versus vehicle group by Student's t test).

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We previously detected QOPs as lineage-committed osteoclast precursors in vivo, and QOPs were isolated from bone marrow as RANK-positive cells.17 We then examined whether ED-71 administration affected the RANK-positive cell population in bone marrow by using FACS (Fig. 4A). The percentage of RANK-positive cells in bone marrow cells obtained from mice treated with ED-71 for 2 and 4 weeks was not significantly different from that of the control mice (Fig. 4A). QOPs differentiated into osteoclasts without cell cycle progression. Hydroxyurea, an inhibitor of DNA replication, inhibits RANKL-induced osteoclastic differentiation from M-CSF-induced bone marrow macrophages but not from QOPs.17 Osteoclast formation using bone marrow cells freshly isolated from mice treated with ED-71 or vehicle for 2 weeks was examined in the presence of hydroxyurea (in vitro QOP assay, Fig. 4B). Similar numbers of TRAP-positive osteoclasts were formed in the presence and absence of ED-71 in the QOP assay. Monocytes/macrophage lineage cells such as F4/80-positive cells and c-Fms-positive cells were shown to be precursors of QOPs. Bone marrow cells obtained from tibias of mice treated with ED-71 and vehicle for 2 weeks were subjected to a FACS analysis using antibodies against monocytes/macrophages (F4/80, c-Fms, CD11b) (Fig. 4C). Populations of F4/80-positive cells, c-Fms-positive cells, and CD11b-positive cells remained unchanged in bone marrow under the ED-71-treated condition (Fig. 4C). These results suggest that the administration of ED-71 does not affect the cellularity of osteoclast precursors in bone marrow in vivo.

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Figure 4. Effects of ED-71 administration on cellularity of osteoclast precursors in bone marrow in mice. ED-71 (50 ng/kg body weight) or vehicle was administered daily to 9-week-old male mice for 2 and 4 weeks. (A) Bone marrow cells obtained from mice treated with ED-71 or vehicle were subjected to a FACS analysis for RANK-positive cells. Percentages of RANK-positive cells among bone marrow cells are provided. (B) QOP in vitro assay. Bone marrow cells obtained from tibias of mice treated with ED-71 or vehicle for 2 weeks were cultured with M-CSF (104 units/mL) and RANKL (5 nmol/L) together with hydroxyurea (50 µmol/L). After 4 days, TRAP-positive cells containing more than three nuclei were counted as osteoclasts. (C) Bone marrow cells were obtained from femurs of mice treated with ED-71 or vehicle for 2 weeks and subjected to a FACS analysis using antibodies against monocyte/macrophage markers (F4/80, c-Fms, 2CD11b). The numbers of F4/80-positive cells, c-Fms-positive cells, and CD11b-positive cells in ED-71-treated mice were expressed as ratios to those in vehicle-treated mice. Values represent the mean ± SEM (n = 3 for each treatment group).

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We next examined the effects of ED-71 on the mRNA expression of osteoclastogenesis-associated molecules (Fig. 5). Tibias were subjected to a quantitative real-time PCR analysis (Fig. 5A). The expression of RANKL mRNA was significantly lower in the ED-71-treated mice than the vehicle-treated mice. ED-71 administration affected neither OPG mRNA nor M-CSF mRNA expression at the two time points of the treatment. Using an immunohistochemical technique, we attempted to identify RANKL-positive cells in bone tissues (Fig. 5B). RANKL-positive cells were observed in the hypertrophic cartilage area and around trabecular and cortical bones. ED-71 administration significantly decreased the number of RANKL-positive cells preferentially around trabecular bones (Fig. 5B, lower panels). When RANKL-positive cell surface (RANKLS) along the trabecular bone and cortical bone (RANKLS/BS, µm/µm), RANKLS along the cortical bone was not significantly affected by ED-71 (Fig. 5C). In contrast, RANKLS along the trabecular bone was significantly decreased in the ED-71-treated mice (Fig. 5C). These results suggest that ED-71 administration preferentially suppressed RANKL expression in osteoblast-lineage cells around the trabecular bones.

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Figure 5. Effects of ED-71 administration on RANKL expression in bone in mice. ED-71 (50 ng/kg body weight) or vehicle was administered daily to 9-week-old male mice for 2 and 4 weeks. (A) Total RNA was prepared from the tibias of mice treated with ED-71 or vehicle and subjected to a quantitative real-time PCR analysis for RANKL, OPG, and M-CSF mRNA. The results for ED-71-treated mice were expressed as a ratio to the value for the vehicle-treated mice. (B) The tibial sections prepared from mice treated with ED-71 or vehicle for 4 weeks were stained with anti-RANKL antibody (green) and DAPI (nuclei, blue). Arrowheads indicate RANKL-positive cells. (C) RANKL-positive cell surface (RANKLS)/bone surface (BS) was measured along the cortical bone and the trabecular bone in mice treated with ED-71 or vehicle for 4 weeks. Values represent the mean ± SEM (n = 3 for each treatment group; *p < 0.05 versus vehicle group by Student's t test).

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We finally examined the effects of ED-71 administration on bone metabolism in OVX mice compared with those of 1α,25(OH)2D3 (Fig. 6). ED-71 (50 ng/kg) or 1α,25(OH)2D3 (50 ng/kg) was administered orally to OVX mice, 5 days a week for 4 weeks. The size of uterus in OVX mice was smaller than that in Sham mice at sacrifice. The body weight of vehicle-treated OVX mice was significantly higher than that of the vehicle-treated Sham mice (OVX 22.0 ± 0.28 g, Sham 20.4 ± 0.17 g, mean ± SD p < 0.01). Trabecular BMD but not cortical BMD in femurs was decreased in OVX mice compared with Sham mice, although the difference was not significant (Fig. 6A). ED-71 administration significantly increased trabecular BMD of OVX mice. Administration of 1α,25(OH)2D3 showed a tendency to increase trabecular BMD, but this increment was not significant. As reported for OVX rats,42 TRAP5b levels of OVX mice were decreased compared with Sham mice (Fig. 6B). Serum levels of TRAP5b and ALP in OVX mice were suppressed significantly by ED-71 and 1α,25(OH)2D3. To verify that bone resorption was enhanced by OVX and that ED-71 suppressed it, serum levels of C-telopeptide of type I collagen (CTX), a bone resorption marker, were determined in those mice (Supplemental Fig. S1). Serum levels of CTX were increased in OVX mice, and the elevated CTX levels were suppressed by ED-71 and 1α,25(OH)2D3. OVX mice showed the enhanced expressions of RANKL and OPG mRNA in bone tissue, both of which were significantly suppressed by ED-71 (Fig. 6C). RANKLS/BS along the trabecular bone and cortical bone was increased in OVX mice (Fig. 6D). ED-71 administration significantly decreased it in trabecular bone but not cortical bone in OVX mice. Overall, the degree of suppression of bone resorption by ED-71 was greater than by 1α,25(OH)2D3 in OVX mice.

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Figure 6. Effects of ED-71 administration on bone resorption in OVX mice. ED-71 (50 ng/kg body weight), 1α,25(OH)2D3 (50 ng/kg body weight), or vehicle was administered to 12-week-old Sham mice and OVX mice 5 days a week for 4 weeks. (A) BMDs of trabecular and cortical bone were measured in the distal femurs of Sham mice treated with vehicle and OVX mice treated with vehicle, ED-71, or 1α,25(OH)2D3. (B) Serum concentrations of TRAP5b and ALP activity were measured in Sham and OVX mice. (C) Total RNA was prepared from the tibias of Sham and OVX mice and subjected to a quantitative real-time PCR analysis for RANKL and OPG mRNA. The results were expressed as a ratio to the value for the vehicle-treated OVX mice. (D) RANKL-positive cell surface (RANKLS)/bone surface (BS) was measured along the trabecular and cortical bone in Sham and OVX mice. Values represent the means ± SEM (n = 3 to 6 for each treatment group; *p < 0.05 versus Sham mice, **p < 0.05 versus vehicle-treated OVX mice by Student's t test).

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Discussion

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

In the current study, we examined the effect on bone metabolism of the daily administration of ED-71 in normal and OVX mice. The treatment increased BMD in the femoral metaphysis with the suppression of both bone resorption and bone formation. It is therefore clear that the increase in BMD was mainly because of the suppression of bone resorption. Bone resorption is known to be tightly coupled to bone formation. The decrease in bone formation caused by ED-71 may be related to the coupling reaction induced by the suppression of bone resorption.

There are two possible explanations for the suppression of bone resorption by ED-71: the first is that ED-71 decreases the number of QOPs, and the second is that ED-71 alters the microenvironment that regulates osteoclastogenesis. Our results support the second possibility. ED-71 affected neither the number of RANK-positive cells (QOPs) in bone marrow nor the osteoclastic differentiation in hydroxyurea-treated bone marrow cultures. It has been reported that monocyte/macrophage-lineage cells such as F4/80-positive cells and c-Fms-positive cells differentiate into QOPs in cultures.17 The number of F4/80-positive cells, c-Fms-positive cells, and CD11b-positive cells remained unchanged in bone marrow even by the long-term treatment with ED-71. RANK-positive cells were detected immunohistochemically along the bone surfaces, and their number was decreased in response to the daily administration of ED-71. These results suggest that most of the immunostainable RANK-positive cells on bone are osteoclasts but not QOPs in mice treated with ED-71 or vehicle. Thus, it was concluded that the number of osteoclasts was decreased, whereas the number of osteoclast precursors including QOPs was not decreased by the daily administration of ED-71.

The daily administration of ED-71 decreased RANKL mRNA expression in bone and reduced the perimeter of RANKLS in trabecular bone. This reduction coincided with a significant decrease in osteoclast numbers in trabecular bone. Although the in vivo characterization of RANKL-positive cells in bone remains to be elucidated, the RANKL-positive cells on bone surfaces undoubtedly belong to the osteoblast lineage cells.

Osteoblast-lineage cells in culture express RANKL in response to 1α,25(OH)2D3.16 ED-71 and 1α,25(OH)2D3 similarly enhanced the expression of RANKL mRNA in osteoblastic cells in dose-dependent manners (Supplemental Fig. S2). Why does the daily administration of ED-71 decrease RANKL-expressing cells in vivo? Several explanations are conceivable. The administration of ED-71 may directly affect osteoblasts to suppress RANKL expression. However, this possibility is unlikely because administering a large amount of ED-71 to normal mice induced hypercalcemia with an increase in osteoclastic bone resorption (data not shown). It has also been reported that administration of an excess amount of ED-71 to OVX rats induces hypercalcemia.43 The inhibitory effect of ED-71 on RANKL expression in osteoblastic cells is not observed in in vitro cultures. These results suggest that long-term exposure to pharmacological doses of ED-71 is required for the suppression of bone resorption in vivo. Osteoclastic differentiation occurs in the area between blood vesicles and bone surfaces, where reticular stromal cells exist.44, 45 Such stromal cells could express RANKL strongly.9 Atkins and colleagues46 reported that RANKL was expressed preferentially by immature osteoblasts and the expression level decreased during their maturation. The daily administration of ED-71 may influence the differentiation of those osteoblast-lineage cells, and this possibility appears likely.

Recently, Nakashima and colleagues47 and Xiong and colleagues48 independently reported that osteocytes are the major source of RANKL in bone remodeling in vivo. Using a series of Cre-deleted strains, they also showed that RANKL expression in the osteoblast-lineage cells is exclusively important for osteoclastogenesis.47, 48 These findings support the possibility that ED-71 may suppress RANKL expression in osteocytes, fully differentiated osteoblasts. However, our experiments showed that ED-71 administration preferentially suppressed the RANKL expression on the surface of trabecular bones. The expression of RANKL on the bone surface appeared to be stronger than that inside the bone tissues. Further studies are necessary to identify the target cells for ED-71 in bone tissue.

The turnover of trabecular bone is much faster than that of cortical bone. The differentiation of osteoblast precursors into mature osteoblasts occurs much more rapidly in trabecular bone than in cortical bone. If the long-term exposure of osteoblast-lineage cells to ED-71 affects their maturation, the effect on osteoblast-lineage cells would be more evident in the trabecular bone. OVX enhances osteoclastic bone resorption, which mainly occurs in the trabecular bone.49 It has been shown that the expression of the RANKL protein is increased during the first stage of maturation in human primary osteoblasts.50 de Freitas and colleagues 43 recently reported that the daily administration of ED-71 to OVX rats enhanced differentiation of preosteoblasts into mature osteoblasts, and suppressed osteoclastic bone resorption. They also showed that apoptosis of osteoclasts in OVX rats was not enhanced by ED-71 administration. Consistent with their findings, ED-71 did not appear to induce apoptosis of osteoclasts but suppressed bone resorption in OVX mice. Taken together, these findings suggest that the long-term exposure to pharmacological doses of ED-71 alters the process of osteoblastic differentiation. Studies are now in progress in our laboratories to elucidate the mechanism by which the administration of ED-71 in vivo influences osteoblast differentiation.

Recent studies have shown that osteoblast-lineage cells support the maintenance of hematopoietic stem cells. CXCL12-abundant reticular (CAR) cells existing around the blood vessels in trabecular bone support differentiation of hematopoietic cells.51 In our experiments, the number of monocytes/macrophages as well as RANK-positive cells remained unchanged in mice treated with ED-71. ED-71 administration affected neither T cells (CD4-positive cells and CD8-positive cells) nor B cells (CD19-positive cells, B220-positive cells and IgM-positive cells) in bone marrow (data not shown). These results suggest that ED-71 administration showed no adverse effect on hematopoietic cell development.

1α,25(OH)2D3 stimulated osteoclast formation in cocultures of osteoblasts and bone marrow cells in a dose-dependent manner.52 ED-71 also stimulated osteoclast formation in vitro in cocultures of osteoblasts and bone marrow cells (Supplemental Fig. S3). In contrast, consistent with the previous findings on 1α,25(OH)2D3,53, 54 ED-71 inhibited RANKL-induced osteoclastic differentiation from precursor cells in the absence of osteoblasts (Supplementary Fig. S3). The efficacy of ED-71 was equivalent to 1α,25(OH)2D3. 1α,25(OH)2D3 has been shown to suppress the expression of c-Fos, a transcription factor essential for osteoclastogenesis, in osteoclast precursors.53 It is also reported that 1α,25(OH)2D3 stimulated the expression of interferon β, an inhibitor of osteoclastogenesis, in osteoclast precursors.54 These results may indicate that vitamin D compounds also act on osteoclast precursors in addition to osteoblast-lineage cells to inhibit osteoclastogenesis in vivo.

Using OVX model mice, we have shown that ED-71 was more potent than 1α,25(OH)2D3 in increasing trabecular BMD. A phase III study in patients with osteoporosis showed that ED-71 increased BMD and reduced the incidence of bone fracture with a greater efficacy than 1α(OH)D3 (alfacalcidol).55 ED-71 has a higher affinity for serum DBP than 1α,25(OH)2D3.36 As a result, ED-71 has a longer half-life than 1α,25(OH)2D3 in plasma.36 This characteristic feature of ED-71 may be related to the finding that ED-71 showed a stronger activity than other active vitamin D compounds in suppressing bone resorption in vivo.

In conclusion, daily administration of ED-71 inhibits osteoclastic differentiation by suppressing RANKL expression in osteoblasts. Our findings raise the possibility that the administration of ED-71 in vivo reduces the number of RANKL-producing osteoblasts, and this possibility is currently being explored in our laboratories.

Disclosures

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

This study has been performed with the financial support of Chugai Pharmaceutical Co., Ltd. SH, SS, ST, HS, and FT are employees of Chugai Pharmaceutical Co., Ltd. HY is an employee of Oriental Yeast Co. All the other 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
  10. Supporting Information

Authors' roles: SH and TM contributed equally to this work. Study design: SH, TM, FT, HS, and NT. Study conduct: NT. Data collection: SH, TM, ST, and SS. Data analysis: SH, TM, and SS. Data interpretation: SH, TM, YK, YN, FT, HS, NU, TS, and NT. Drafting manuscript: SH and TM. Revising manuscript content: YK, YN, NU, TS, and NT. Approving final version of manuscript: SH, TM, YK, YN, ST, SS, FT, HS, HY, NU, TS, and NT. NT takes responsibility for the integrity of the data analysis. HY provided GST-RANKL.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

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

Additional Supporting Information may be found in the online version of this article.

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