Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP.

Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135-210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173-210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS-PAGE, dynAP migrates anomalously at ~ 45 kDa, much larger than expected (22.5 kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135-210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.

Characterization, pharmacokinetics, and pharmacodynamics of anti-Siglec-15 antibody and its potency for treating osteoporosis and as follow-up treatment after parathyroid hormone use.

Recent studies have established the idea that Siglec-15 is involved in osteoclast differentiation and/or function, and it is anticipated that therapies suppressing Siglec-15 function can be used to treat bone diseases such as osteoporosis. We have produced rat monoclonal anti-Siglec-15 antibody (32A1) and successively generated humanized monoclonal anti-Siglec-15 antibody (DS-1501a) from 32A1. Studies on the biological properties of DS-1501a showed its specific binding affinity to Siglec-15 and strong activity to inhibit osteoclastogenesis. 32A1 inhibited multinucleation of osteoclasts and bone resorption (pit formation) in cultured mouse bone marrow cells. 32A1 also inhibited pit formation in cultured human osteoclast precursor cells. Maximum serum concentration and serum exposure of DS-1501a in rats were increased in a dose-dependent manner after single subcutaneous or intravenous administration. Furthermore, single administration of DS-1501a significantly suppressed bone resorption markers with minimal effects on bone formation markers and suppressed the decrease in bone mineral density (BMD) of the lumbar vertebrae in ovariectomized (OVX) rats. In histological analysis, the osteoclasts distant from the chondro-osseous junction of the tibia tended to be flattened, shrunken, and functionally impaired in 32A1-treated rats, while alkaline phosphatase-positive osteoblasts were observed throughout the metaphyseal trabeculae. In addition, we compared the efficacy of 32A1 with that of alendronate (ALN) as follow-up medicine after treatment with parathyroid hormone (PTH) using mature established osteoporosis rats. The beneficial effect of PTH on bone turnover disappeared 8 weeks after discontinuing the treatment. The administration of 32A1 once every 4 weeks for 8 weeks suppressed bone resorption and bone formation when the treatment was switched from PTH to 32A1, leading to the maintenance of BMD and bone strength. Unlike with ALN, the onset of suppression of bone resorption with 32A1 was rapid, while the suppression of bone formation was mild. The improvement of bone mass, beneficial bone turnover balance, and suppression of osteoclast differentiation/multinucleation achieved by 32A1 were supported by histomorphometry. Notably, the effects of 32A1 on bone strength, not only structural (extrinsic) but also material (intrinsic) properties, were significantly greater than those of ALN. Since the effect of 32A1 on BMD was moderate, its effect on bone strength could not be fully explained by the increase in BMD. The beneficial balance of bone turnover caused by 32A1 might, at least in part, be responsible for the improvement in bone quality. This is the first report describing the effects of anti-Siglec-15 antibody in OVX rats; the findings suggest that this antibody could be an excellent candidate for treating osteoporosis, especially in continuation therapy after PTH treatment, due to its rapid action and unprecedented beneficial effects on bone quality.

Anti-Siglec-15 antibody suppresses bone resorption by inhibiting osteoclast multinucleation without attenuating bone formation.

Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.

Discovery of DS79932728: A Potent, Orally Available G9a/GLP Inhibitor for Treating ß-Thalassemia and Sickle Cell Disease.

Therapeutic reactivation of the γ-globin genes for fetal hemoglobin (HbF) production is an attractive strategy for treating ß-thalassemia and sickle cell disease. It was reported that genetic knockdown of the histone lysine methyltransferase EHMT2/1 (G9a/GLP) is sufficient to induce HbF production. The aim of the present work was to acquire a G9a/GLP inhibitor that induces HbF production sufficiently. It was revealed that tetrahydroazepine has versatility as a side chain in various skeletons. We ultimately obtained a promising aminoindole derivative (DS79932728), a potent and orally bioavailable G9a/GLP inhibitor that was found to induce γ-globin production in a phlebotomized cynomolgus monkey model. This work could facilitate the development of effective new approaches for treating ß-thalassemia and sickle cell disease.

Osteoclast differentiation by RANKL and OPG signaling pathways.

INTRODUCTION: In bone tissue, bone resorption by osteoclasts and bone formation by osteoblasts are repeated continuously. Osteoclasts are multinucleated cells that derive from monocyte-/macrophage-lineage cells and resorb bone. In contrast, osteoblasts mediate osteoclastogenesis by expressing receptor activator of nuclear factor-kappa B ligand (RANKL), which is expressed as a membrane-associated cytokine. Osteoprotegerin (OPG) is a soluble RANKL decoy receptor that is predominantly produced by osteoblasts and which prevents osteoclast formation and osteoclastic bone resorption by inhibiting the RANKL-RANKL receptor interaction. MATERIALS AND METHODS: In this review, we would like to summarize our experimental results on signal transduction that regulates the expression of RANKL and OPG. RESULTS: Using OPG gene-deficient mice, we have demonstrated that OPG and sclerostin produced by osteocytes play an important role in the maintenance of cortical and alveolar bone. In addition, it was shown that osteoclast-derived leukemia inhibitory factor (LIF) reduces the expression of sclerostin in osteocytes and promotes bone formation. WP9QY (W9) is a peptide that was designed to be structurally similar to one of the cysteine-rich TNF-receptortype-I domains. Addition of the W9 peptide to bone marrow culture simultaneously inhibited osteoclast differentiation and stimulated osteoblastic cell proliferation. An anti-sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) antibody inhibited multinucleated osteoclast formation induced by RANKL and macrophage colony-stimulating factor (M-CSF). Pit-forming activity of osteoclasts was also inhibited by the anti-Siglec-15 antibody. In addition, anti-Siglec-15 antibody treatment stimulated the appearance of osteoblasts in cultures of mouse bone marrow cells in the presence of RANKL and M-CSF. CONCLUSIONS: Bone mass loss depends on the RANK-RANKL-OPG system, which is a major regulatory system of osteoclast differentiation induction, activation, and survival.

Discovery of novel histone lysine methyltransferase G9a/GLP (EHMT2/1) inhibitors: Design, synthesis, and structure-activity relationships of 2,4-diamino-6-methylpyrimidines.

The discovery and optimization of a novel series of G9a/GLP (EHMT2/1) inhibitors are described. Starting from known G9a/GLP inhibitor 5, efforts to explore the structure-activity relationship and optimize drug properties led to a novel compound 13, the side chain of which was converted to tetrahydroazepine. Compound 13 showed increased G9a/GLP inhibitory activity compared with compound 5. In addition, compound 13 exhibited improved human ether-a-go-go related gene (hERG) inhibitory activity over compound 5 and also improved pharmacokinetic profile in mice (oral bioavailability: 17 to 40%). Finally, the co-crystal structure of G9a in complex with compound 13 provides the basis for the further development of tetrahydroazepine-based G9a/GLP inhibitors.

Siglec-15-targeting therapy protects against glucocorticoid-induced osteoporosis of growing skeleton in juvenile rats.

Effective treatment of juvenile osteoporosis, which is frequently caused by glucocorticoid (GC) therapy, has not been established due to limited data regarding the efficacy and adverse effects of antiresorptive therapies on the growing skeleton. We previously demonstrated that sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) targeting therapy, which interferes with osteoclast terminal differentiation in the secondary, but not primary, spongiosa, increased bone mass without adverse effects on skeletal growth, whereas bisphosphonate, a first-line treatment for osteoporosis, increased bone mass but impaired long bone growth in healthy growing rats. In the present study, we investigated the efficacy of anti-Siglec-15 neutralizing antibody (Ab) therapy against GC-induced osteoporosis in a growing rat model. GC decreased bone mass and deteriorated mechanical properties of bone, due to a disproportionate increase in bone resorption. Both anti-Siglec-15 Ab and alendronate (ALN) showed protective effects against GC-induced bone loss by suppressing bone resorption, which was more pronounced with anti-Siglec-15 Ab treatment, possibly due to a reduced negative impact on bone formation. ALN induced histological abnormalities in the growth plate and morphological abnormalities in the long bone metaphysis but did not cause significant growth retardation. Conversely, anti-Siglec-15 Ab did not show any negative impact on the growth plate and preserved normal osteoclast and chondroclast function at the primary spongiosa. Taken together, these results suggest that anti-Siglec-15 targeting therapy could be a safe and efficacious prophylactic therapy for GC-induced osteoporosis in juvenile patients.

Siglec-15-targeting therapy increases bone mass in rats without impairing skeletal growth.

The treatment of juvenile osteoporosis has not been established due to a lack of data regarding the efficacy and adverse effects of therapeutic agents. The possible adverse effects of the long-term use of antiresorptive therapies on skeletal growth in children is of particular concern. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is an immunoreceptor that regulates osteoclast development and bone resorption, and its deficiency suppresses bone remodeling in the secondary spongiosa, but not in the primary spongiosa, due to a compensatory mechanism of osteoclastogenesis. This prompted us to develop an anti-Siglec-15 therapy for juvenile osteoporosis because most anti-resorptive drugs have potential adverse effects on skeletal growth. Using growing rats, we investigated the effects of an anti-Siglec-15 neutralizing antibody (Ab) on systemic bone metabolism and skeletal growth, comparing this drug to bisphosphonate, a first-line treatment for osteoporosis. Male 6-week-old F344/Jcl rats were randomized into six groups: control (PBS twice per week), anti-Siglec-15 Ab (0.25, 1, or 4 mg/kg every 3 weeks), and alendronate (ALN) (0.028 or 0.14 mg/kg twice per week). Treatment commenced at 6 weeks of age and continued for the next 6 weeks. Changes in bone mass, bone metabolism, bone strength, and skeletal growth during treatment were analyzed. Both anti-Siglec-15 therapy and ALN increased bone mass and the mechanical strength of both the femora and lumbar spines in a dose-dependent manner. Anti-Siglec-15 therapy did not have a significant effect on skeletal growth as evidenced by micro-CT-based measurements of femoral length and histology, whereas high-dose ALN resulted in growth retardation with histological abnormalities in the growth plates of femurs. This unique property of the anti-Siglec-15 Ab can probably be attributed to compensatory signaling for Siglec-15 inhibition in the primary spongiosa, but not in the secondary spongiosa. Thus, anti-Siglec-15 therapy could be a safe and effective for juvenile osteoporosis.

Impaired osteoclast differentiation and function and mild osteopetrosis development in Siglec-15-deficient mice.

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a cell surface receptor for sialylated glycan ligands. Recent in vitro studies revealed upregulated Siglec-15 expression in differentiated osteoclasts and inhibition of osteoclast differentiation by anti-Siglec-15 polyclonal antibody, demonstrating Siglec-15 involvement in osteoclastogenesis. To discern the physiological role of Siglec-15 in skeletal development and osteoclast formation and/or function in vivo, we generated Siglec-15-deficient (siglec-15(-/-)) mice and analyzed their phenotype. The siglec-15(-/-) mice developed without physical abnormalities other than increased trabecular bone mass in lumbar vertebrae and metaphyseal regions of the femur and tibia, causing mild osteopetrosis. Histological analyses demonstrated that the number of osteoclasts present on the femoral trabecular bone of the mutant mice was comparable to that of the wild-type mice. However, urinary deoxypyridinoline, a systemic bone resorption marker, decreased in the siglec-15(-/-) mice, indicating that impaired osteoclast function was responsible for increased bone mass in the mutant mice. In addition, the ability of bone marrow-derived monocytes/macrophages from the siglec-15(-/-) mice to differentiate into osteoclasts was impaired, as determined in vitro by cellular tartrate-resistant acid phosphatase activity in response to the receptor activator of nuclear factor-κB ligand or tumor necrosis factor-α. These results reveal the importance of Siglec-15 in the regulation of osteoclast formation and/or function in vivo, providing new insights into osteoclast biology.

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation.

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D(3) and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

PEGylation of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) results in decreased uptake into rats and human liver.

OBJECTIVES: Our aim was to investigate the effect of PEGylation on the uptake of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) into rat liver, kidney and spleen, and human liver. METHODS: Copolymer of polyethyleneglycol allylmethylether and maleamic acid sodium salt with OCIF (poly(PEG)-OCIF) (0.5 mg/kg) was administered to rats and the concentrations of poly(PEG)-OCIF in the liver, kidney and spleen at 15 min after administration were measured by ELISA. For human liver uptake, the liver perfusion of OCIF and (3)H-labelled poly(PEG)-OCIF was conducted using fresh human liver block. KEY FINDINGS: The tissue uptake of poly(PEG)-OCIF in rats was significantly lower compared with that of OCIF. In fresh human liver perfusion, (3)H-poly(PEG)-OCIF was rarely taken up into the liver. On the other hand, more than 50% of the perfused OCIF was taken up. CONCLUSIONS: PEGylation of OCIF using poly(PEG) dramatically suppressed the uptake of OCIF into human liver as well as into rat liver and could be a promising approach for improving the pharmacokinetic and pharmacological effects of OCIF in the clinical setting.

Vitamin K2 inhibits glucocorticoid-induced bone loss partly by preventing the reduction of osteoprotegerin (OPG).

We have recently demonstrated that glucocorticoid (GC) suppresses bone formation and enhances bone resorption, with resultant bone loss. This altered bone turnover is not due to the action of parathyroid hormone (PTH), but appears to be related to the suppression of osteoprotegerin (OPG). As vitamin K2 (menatetrenone) has been used for the treatment of osteoporosis, the present study was carried out to evaluate the effect of vitamin K2 on GC-induced bone loss. Twenty patients with chronic glomerulonephritis treated with GC for the first time were chosen for this study. Ten patients received GC alone (group A) and the other 10 patients each received 15 mg of vitamin K2 per day in addition to GC (group B). Markers of bone metabolism, including serum OPG, osteocalcin (OC), bone-specific alkaline phosphatase activity (BAP), PTH, tartrate-resistant acid phosphatase (TRAP), and bone mineral density (BMD), were measured before and during the treatment. OPG was significantly decreased in group A (P < 0.001), while no significant change was seen in group B. TRAP was markedly increased in both groups, more particularly in group A (P < 0.01). PTH was decreased in group A, but was increased in group B. OC was decreased at month 1 but subsequently increased until month 12 in both groups. BAP had decreased at month 3 in group A (P < 0.05), but not in group B. BMD of the lumbar spine was significantly reduced after 6 months (P < 0.01), and 12 months (P < 0.001) of treatment in group A, whereas there was no remarkable change in group B. The present study demonstrated that the inhibition exerted by vitamin K2 of the reduction in OPG induced by GC may, at least in part, play a role in the prevention and treatment of GC-induced bone loss.

Transgenic mice overexpressing soluble osteoclast differentiation factor (sODF) exhibit severe osteoporosis.

Osteoclast differentiation factor, ODF, also called RANKL, TRANCE, or OPGL, is a key molecule for osteoclast differentiation and activation, and is thought to act as a membrane-associated molecule in bone remodeling. Recent study suggested that soluble ODF (sODF) released from T cells also has some roles in bone resorption. To investigate the physiological and pathological function of sODF, we generated two types of transgenic mice overexpressing sODF. Mice overexpressing sODF ubiquitously from the early developmental stage died at the late fetal stage. The other type of mice, expressing sODF only in the liver after birth, grew to maturity with normal body size and weight. However, they exhibited a marked decrease in bone mineral density with aging compared with their non-transgenic littermates, and in addition, the strength of their femurs was extremely reduced. Histological analysis showed that the trabecular bone mass was decreased at 6 weeks of age and was sparse at age 3-4 months. The number of osteoclasts was significantly increased, while the number of osteoblasts was not altered on the surface of young trabecular bone. These results indicate that excessive production of sODF causes osteoporosis by accelerated osteoclastogenesis. The transgenic mouse overexpressing sODF in the liver could serve as a useful animal model for studying bone remodeling and evaluating therapeutic agents for osteoporosis.

Increased circulating levels of osteoclastogenesis inhibitory factor (osteoprotegerin) in patients with chronic renal failure.

Skeletal resistance to parathyroid hormone (PTH) is one of the major abnormalities underlying bone diseases in uremia, the mechanism of which has not yet been fully elucidated. Osteoclastogenesis inhibitory factor (OCIF), or osteoprotegerin, is a natural decoy receptor for osteoclast differentiation factor (ODF), produced by osteoblasts in response to PTH. To elucidate the kinetics and roles of OCIF in chronic renal failure, serum OCIF levels were measured in 46 predialysis patients and 21 dialysis patients by means of enzyme-linked immunosorbent assay (ELISA). Serum OCIF levels in predialysis patients increased as renal function declined (OCIF = 1.178 + 0.233 x creatinine; r2 = 0.413; P < 0.0001). Twenty-four-hour creatinine clearance and 1/OCIF in predialysis patients showed a clear positive correlation and a straight line regression (1/OCIF = 0.443 + 0.004 x creatinine clearance; r2 = 0.425; P < 0.0001). In dialysis patients, serum OCIF levels were significantly elevated (5.18 +/- 1.48 ng/mL) to a level that would inhibit 50% osteoclast formation in vitro. These findings suggest that OCIF accumulates in serum of patients with renal dysfunction. Because serum levels of OCIF with the ability to bind ODF in vitro (active OCIF) correlated well with those of OCIF detected by standard ELISA (active OCIF = 0.251 + 0.877 x OCIF; r2 = 0.829; P < 0.0001), OCIF accumulated in serum may be a candidate uremic toxin responsible for the skeletal resistance to PTH seen in chronic renal failure. Further studies with serum parameters and bone histological evaluation are needed to assess this possibility.

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis. III. Immunosuppressive efficacy.

Novel immunosuppressants, SNF4435C and D produced by a strain of Streptomyces spectabilis, were examined for their pharmacodynamical profiles. SNF4435C and D suppressed the responses of both murine splenocytes and human peripheral blood lymphocytes in the mixed lymphocyte reaction (MLR) with IC50 values of 0.5 microM and 0.2 microM, respectively. In the mouse MLR experiments, SNF4435C and D did not block the production of interleukin-2 (IL-2) and the compounds-induced suppression was not restored by the addition of exogeneous IL-2. In addition, the significant inhibitory action was still retained even when SNF4435C or D was added after 48 hours from the start of the culture. These results were distinct from the behaviors observed with FK-506. SNF4435C, the major component, suppressed mouse delayed type hypersensitivity (DTH) and prolonged rat skin allograft survival.

Osteoclastogenesis inhibitory factor/osteoprotegerin ameliorates the decrease in both bone mineral density and bone strength in immobilized rats.

Rat models of immobilization-induced osteopenia are characterized by uncoupling of bone metabolism, i.e., increased bone resorption and decreased bone formation in trabecular bone. Using such a rat model, the efficacy of osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin, a novel secreted protein that inhibits osteoclastogenesis, in reducing bone loss was investigated. Male Fischer rats were neurectomized and injected intramuscularly with either OCIF (0.2, 1.0, or 5.0 mg/kg body weight) or vehicle once daily for 7 days. On the eighth day after sciatic neurectomy, significant bone loss was observed in the vehicle-injected rats. OCIF ameliorated the decrease in bone mineral density (BMD) of both the proximal and distal femur in a dose-dependent manner. OCIF also ameliorated the decrease in bone strength of the femoral neck at the highest dose. A high correlation (r = 0.805) was detected between the BMD of the distal femur and the bone strength of the femoral neck. When OCIF was administered intermittently to the immobilized rats twice weekly (on days 1 and 4) after immobilization, it also ameliorated the decrease in BMD of the distal femur. These results suggest that OCIF has therapeutic potential for the treatment of immobilization-induced osteopenia.

[Osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG)].

Osteoclastogenesis inhibitory factor (OCIF) was isolated from the conditioned medium of human embryonic lung fibroblasts. OCIF is a novel member of the tumor necrosis factor receptor superfamily and identical with Osteoprotegerin (OPG) discovered by the Amgen researchers. Consequently, through the identification of receptor activator of NF-kappaB ligand (RANKL) as a target molecule of OCIF/OPG, it was demonstrated that RANKL is a crucial factor in the differentiation, maturation, and activation of osteoclasts. Discovery of OCIF/OPG and RANKL has broken new ground in the field of bone physiopathology. Moreover, OCIF/OPG not only contributes to the field of basic science but also is anticipated as a novel therapeutic candidate for the treatment of primary and secondary osteoporosis through a series of pre-clinical and clinical studies. Because OCIF/OPG and RANKL have been proven to be involved in the onset and development of many metabolic bone diseases besides osteoporosis, OCIF/OPG is also expected as a therapeutic candidate for the treatment of such bone diseases.