ABSTRACT
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.
Subject(s)
Bone Resorption , Osteoporosis , Alendronate/pharmacology , Animals , Bone Density , Bone Resorption/drug therapy , Female , Follow-Up Studies , Humans , Immunoglobulins/pharmacology , Membrane Proteins , Mice , Osteoporosis/drug therapy , Ovariectomy , Parathyroid Hormone/pharmacology , Parathyroid Hormone/therapeutic use , Rats , Sialic Acid Binding Immunoglobulin-like Lectins/pharmacologyABSTRACT
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.
Subject(s)
Bone Resorption , Osteoporosis , Alendronate/adverse effects , Animals , Bone Resorption/chemically induced , Bone Resorption/drug therapy , Bone Resorption/pathology , Bone and Bones/pathology , Glucocorticoids/adverse effects , Humans , Osteoporosis/chemically induced , Osteoporosis/drug therapy , Osteoporosis/prevention & control , Rats , Sialic Acid Binding Immunoglobulin-like LectinsABSTRACT
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 4â¯mg/kg every 3â¯weeks), and alendronate (ALN) (0.028 or 0.14â¯mg/kg twice per week). Treatment commenced at 6â¯weeks of age and continued for the next 6â¯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.
Subject(s)
Bone Development , Bone and Bones/pathology , Membrane Proteins/antagonists & inhibitors , Molecular Targeted Therapy , Alendronate/pharmacology , Animals , Antibodies/pharmacology , Biomarkers/metabolism , Biomechanical Phenomena/drug effects , Bone Development/drug effects , Bone and Bones/drug effects , Bone and Bones/metabolism , Male , Membrane Proteins/metabolism , Organ Size/drug effects , RatsABSTRACT
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.
Subject(s)
Cell Differentiation , Immunoglobulins/physiology , Membrane Proteins/physiology , Osteoclasts/cytology , Osteopetrosis/pathology , Absorptiometry, Photon , Animals , Bone Density , Female , Immunoglobulins/genetics , Male , Membrane Proteins/genetics , Mice , Mice, KnockoutABSTRACT
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.