Osteoclast Inhibitory Factor Research Articles

Sclerostin vaccination mitigates estrogen deficiency induction of bone mass loss and microstructure deterioration.

Sclerostin (SOST) is a Wnt signaling inhibitor detrimental to osteogenic differentiation and bone mineral acquisition. While control of SOST action delays the pathogenesis of skeletal disorders, the effects of SOST vaccination on the estrogen deficiency-induced bone deterioration remain elusive. In this study, we generated a SOST-Fc fusion protein which was composed of a SOST peptide Pro-Asn-Ala-Ile-Gly along with an IgG Fc fragment. SOST-Fc vaccination increased serum anti-SOST antibody levels and reduced serum SOST concentrations in mice. In vitro, anti-SOST serum attenuated the SOST-induced inhibition of osteogenic gene expression in osteoblast cultures. Administration with SOST-Fc increased serum levels of bone formation marker osteocalcin and alleviated the ovariectomy escalation of serum resorption markers CTX-1 and TRAP5b concentrations. It remarkably lessened the estrogen deficiency-mediated deterioration of bone mineral density, morphometric characteristics of trabecular bone, and mechanical strength of femurs and lumbar spines. The SOST-Fc-treated skeletal tissue exhibited moderate responses to the adverse actions of ovariectomy to bone mineral accretion, osteoclast surface, trabecular separation, and fatty marrow histopathology. SOST-Fc treatment increased serum osteoclast-inhibitory factor osteoprotegrin levels in conjunction with strong Wnt3a, β-catenin, and TCF4 immunostaining in osteoblasts, whereas it weakened the estrogen deficiency enhancement of osteoclast-promoting factor receptor activator of nuclear factor-κB ligand. Taken together, blockade of SOST action by SOST-Fc vaccination sustains Wnt signaling, which harmonizes bone mineral accretion and resorption reactions and thereby ameliorates ovariectomy-induced bone loss. This study highlights SOST-Fc fusion protein as a new molecular therapeutic potential for preventing from osteoporotic disorders.

Inférence causale dans les essais cliniques et la recherche épidémiologique

Sclerostin (SOST) is a Wnt signaling inhibitor detrimental to osteogenic differentiation and bone mineral acquisition. While control of SOST action delays the pathogenesis of skeletal disorders, the effects of SOST vaccination on the estrogen deficiency-induced bone deterioration remain elusive. In this study, we generated a SOST-Fc fusion protein which was composed of a SOST peptide Pro-Asn-Ala-Ile-Gly along with an IgG Fc fragment. SOST-Fc vaccination increased serum anti-SOST antibody levels and reduced serum SOST concentrations in mice. In vitro, anti-SOST serum attenuated the SOST-induced inhibition of osteogenic gene expression in osteoblast cultures. Administration with SOST-Fc increased serum levels of bone formation marker osteocalcin and alleviated the ovariectomy escalation of serum resorption markers CTX-1 and TRAP5b concentrations. It remarkably lessened the estrogen deficiency-mediated deterioration of bone mineral density, morphometric characteristics of trabecular bone, and mechanical strength of femurs and lumbar spines. The SOST-Fc-treated skeletal tissue exhibited moderate responses to the adverse actions of ovariectomy to bone mineral accretion, osteoclast surface, trabecular separation, and fatty marrow histopathology. SOST-Fc treatment increased serum osteoclast-inhibitory factor osteoprotegrin levels in conjunction with strong Wnt3a, β-catenin, and TCF4 immunostaining in osteoblasts, whereas it weakened the estrogen deficiency enhancement of osteoclast-promoting factor receptor activator of nuclear factor-κB ligand. Taken together, blockade of SOST action by SOST-Fc vaccination sustains Wnt signaling, which harmonizes bone mineral accretion and resorption reactions and thereby ameliorates ovariectomy-induced bone loss. This study highlights SOST-Fc fusion protein as a new molecular therapeutic potential for preventing from osteoporotic disorders.

Regulation of bone metabolism by Wnt signals.

Wnt ligands play a central role in the development and homeostasis of various organs through β-catenin-dependent and -independent signalling. The crucial roles of Wnt/β-catenin signals in bone mass have been established by a large number of studies since the discovery of a causal link between mutations in the low-density lipoprotein receptor-related protein 5 (Lrp5) gene and alternations in human bone mass. The activation of Wnt/β-catenin signalling induces the expression of osterix, a transcription factor, which promotes osteoblast differentiation. Furthermore, this signalling induces the expression of osteoprotegerin, an osteoclast inhibitory factor in osteoblast-lineage cells to prevent bone resorption. Recent studies have also shown that Wnt5a, a typical non-canonical Wnt ligand, enhanced osteoclast formation. In contrast, Wnt16 inhibited osteoclast formation through β-catenin-independent signalling. In this review, we discussed the current understanding of the Wnt signalling molecules involved in bone formation and resorption.

CCR5-CCL Axis in PDL during Orthodontic Biophysical Force Application

Tooth movement by application of orthodontic biophysical force primarily reflects the role of soluble molecules released from the periodontal ligament (PDL). Thus far, many factors have been reported to be involved in orthodontic tooth movement (OTM), but key molecules that orchestrate responses of periodontal tissues to biophysical force are still enigmatic. In this in vivo study, in which the upper first molars in rats were moved, differential display–polymerase chain reaction revealed that CC chemokine receptor 5 (CCR5) level was differentially increased during OTM. Strong immunoreactivity for CCR5 was found in the PDL undergoing force application. Moreover, the in vitro compression or tension force application to primary cultured human PDL cells increased the expression of CCR5 and CCR5 ligands. In vitro tension force on human PDL cells did not induce RANKL, an osteoclastogenesis-inducing factor, but did induce the upregulation of IL12, an osteoclast inhibitory factor, and osteoblast differentiation factors, including Runx2, which was attenuated under tension by CCR5 gene silencing whereas augmented with CCR5 ligands. In contrast, in vitro compression force did not induce the expression of osteoprotegerin, a decoy receptor for RANKL and Runx2, but did induce the upregulation of RANKL, which was attenuated under compression by CCR5 gene silencing. These results suggest that the CCR5–CCR5 ligands axis in PDL cells may play a crucial role in the remodeling of periodontal tissues and can be a therapeutic target for achieving efficient OTM.

Effects of Osteochondrin S and select connective tissue ribonucleinate components on human osteoclasts in vitro

Osteochondrin S, a natural product derived from connective tissues and yeast, is used to treat osteoarthritis. The aim of this study was to determine the effect of Osteochondrin S on human osteoclast activity in vitro. Osteoclasts were derived from human peripheral blood mononuclear cells stimulated with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B (RANK) ligand. Cells were treated with 23.5-587.2 ng/ml Osteochondrin S or 0.2-5 mg/ml of RNA components (synovia, placenta, intervertebral disc or cartilage). The effects on osteoclast formation and resorptive activity were assessed. Real-time polymerase chain reaction was conducted to assess the expression of key osteoclast genes. Osteochondrin S and the individual RNA extracts resulted in a concentration-dependent inhibition of human osteoclast activity. Osteochondrin S did not affect RANK, nuclear factor of activated T cells (NFATc1), osteoclast-associated receptor or cathepsin K expression. However, there was a significant (P < 0.05) reduction in mRNA expression of calcitonin receptor. Osteochondrin S treatment also significantly increased the expression of osteoclast inhibitory factor interferon-β and, interestingly, increased the expression of tumour necrosis-α-like weak inducer of apoptosis (TWEAK). Osteochondrin S inhibited the resorptive ability of osteoclasts. These actions are likely to occur at a late stage during osteoclast formation, downstream of NFATc1. Overall, the findings show that Osteochondrin S inhibition of osteoclast activity may be responsible for its beneficial effects on diseases such as osteoarthritis.

Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.

Osteolytic bone resorption in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T lymphotropic virus type 1 (HTLV-1). Patients with ATLL frequently develop humoral hypercalcemia of malignancy (HHM) resulting from increased osteoclastic bone resorption. Our goal was to investigate the mechanisms of ATLL-induced osteoclastic bone resorption. Murine calvaria co-cultured with HTLV-1-infected cells directly or conditioned media from cell cultures had increased osteoclast activity that was dependent on RANKL, indicating that factors secreted from ATLL cells had a stimulatory effect on bone resorption. Factors released from resorbing bone stimulated proliferation of HTLV-1-infected T-cells. Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α), both osteoclast stimulators, were expressed in HTLV-1-infected T-cell lines. Interestingly, when HTLV-1-infected T-cells were co-cultured with pre-osteoblasts, the expression of osteoprotegerin (OPG), an osteoclast inhibitory factor, was significantly down-regulated in the pre-osteoblasts. When OPG was added into the ex vivo osteoclastogenesis assay induced by HTLV-1-infected T-cells, osteoclastogenesis was strongly inhibited. In addition, HTLV-1-infected T-cells inhibited expression of early osteoblast genes and induced late genes. These regulators will serve as future therapeutic targets for the treatments of HHM in ATLL.

Involvement of molecular mimicry between human T‐cell leukemia virus type 1 gp46 and osteoprotegerin in induction of hypercalcemia

Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), frequently associated with hypercalcemia and bone destruction. A positive correlation between the appearance of an antibody recognizing the central region (Asp197 to Leu216) on Gp46, gp46-197, and the severity of ATL has been demonstrated. In this study, five male Nihon Hakusyoku rabbits were immunized with a synthetic peptide corresponding to the gp46-197 region to clarify its action and mechanism. Two of the rabbits showed piloerection, anorexia, and somnolence, and died soon after booster administration. The serum calcium level of the dead rabbits was significantly high, compared to those of surviving rabbits. Interestingly, amino acid sequences homologous with gp46-197 were found in the carboxyl-terminal half of osteoprotegerin (OPG), an osteoclast inhibitory factor. To confirm the effect of the gp46-197 region on osteogenesis in vivo, the peptide was intraperitoneally administered to male Sprague-Dawley rats. The administration of the gp46-197 peptide resulted in a decrease of bone mineral density (BMD), a significant increase of serum calcium level, and inhibition of normal bone growth in both short- and long-term experiments. In rats, femoral growth inhibition by the gp46-197 peptide was restored by the coadministration of recombinant human OPG. Improvement by OPG in the adverse effect indicates that the central region of HTLV-1 Gp46 acts as an antagonist for OPG and leads to hypercalcemia.

HTLV-1 Tax transgenic mice develop spontaneous osteolytic bone metastases prevented by osteoclast inhibition

One in 20 carriers of human T-cell leukemia virus type 1 (HTLV-1) will develop adult T-cell leukemia/lymphoma (ATL), a disease frequently associated with hypercalcemia, bone destruction, and a fatal course refractory to current therapies. Overexpression of the HTLV-1-encoded Tax oncoprotein under the human granzyme B promoter causes large granular lymphocytic leukemia/lymphomas in mice. We found that Tax+ mice spontaneously developed hypercalcemia, high-frequency osteolytic bone metastases, and enhanced osteoclast activity. We evaluated Tax tumors for the production of osteoclast-activating factors. Purification of Tax+ tumor cells and nonmalignant tumor-infiltrating lymphocytes demonstrated that each of these populations expressed transcripts for distinct osteoclast-activating factors. We then evaluated the effect of osteoclast inhibition on tumor formation. Mice doubly transgenic for Tax and the osteoclast inhibitory factor, osteoprotegerin, were protected from osteolytic bone disease and developed fewer soft-tissue tumors. Likewise, osteoclast inhibition with bone-targeted zoledronic acid protected Tax+ mice from bone and soft-tissue tumors and prolonged survival. Tax+ mice represent the first animal model of high-penetrance spontaneous osteolytic bone metastasis and underscore the critical role of nonmalignant host cells recruited by tumor cells in the process of cancer progression and metastasis.

The inhibitor of differentiation-2 promotes synovial fibroblast-dependent osteoclastogenesis in rheumatoid arthritis

Objectives: Despite indirect evidence suggesting that low oxygen levels might occur in the rheumatoid arthritis (RA) synovium, direct proof of the presence of hypoxia in the arthritic synovium as well as the relevance of low oxygen levels for joint destruction is lacking. The aim of this study was to analyse the distribution of hypoxia in arthritic joints and to evaluate the molecular effects of the hypoxic environment on the phenotype of RA synovial fibroblasts (SF). Methods: The hypoxia marker EF-5 was applied in mice with the collagen-induced arthritis (CIA). Expression profile analysis with hypoxic and normoxic SF was performed using subtractive hybridization and microarray. The expression of the inhibitor of differentiation-2 (Id-2), CD68 (macrophage marker) and prolyl hydroxylase (fibroblast marker) was evaluated by immunohistochemistry on synovial tissues from RA, osteoarthritis patients and CIA mice. To evaluate the function of Id-2 in SF, cells were transfected with the pcDNA3.1 containing cDNA for Id-2 or Id-2-specific siRNA or mock controls. The expression of Id-2 and genes regulated by Id-2 in transfected SF was evaluated by SYBR Green real-time PCR and western blot. SF stably transfected with Id-2 were cocultured with bone marrow cells in a transwell system. The expression of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin were measured by real-time PCR. The development of osteoclasts was evaluated by visualization of the activity of tartrate-resistant acid phosphatase. Results: Using the hypoxia marker EF-5 we found that in mice with CIA, synovial cells invading bone and cartilage are exposed to reduced oxygen levels. Expression profile studies identified Id-2 as being upregulated under low oxygen conditions. In addition, IL-1beta stimulation increased the expression of Id-2 in these cells. Histological studies of RA synovium and CIA synovium showed strong expression of Id-2 in SF at sites of synovial invasion into bone. Overproduction of Id-2 in SF by stable transfection triggered the expression of several genes promoting osteoclastogenesis, including BMP-2, PTHrP, Wnt5a and vascular endothelial growth factor. Conversely, the suppression of endogenous Id-2 led to the downregulation of the expression of these molecules. Consistent with these findings coculture of Id-2 transfected SF with bone marrow cells increased the expression of the osteoclast differentiation factor RANKL, and decreased the expression of the osteoclast inhibitory factor osteoprotegerin in bone marrow stromal cells, which was followed by an increase in the number of osteoclasts. Conclusion: Taken together, our data provide evidence that hypoxia is present at sites of synovial invasion in RA and that Id-2 induced by hypoxia contributes at these sites to joint destruction by promoting SF-dependent osteoclastogenesis.

Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic

Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic

Effects of a bisphosphonate on the expression of bone specific genes after autogenous free bone grafting in rats.

The purpose of this study was to evaluate the clinical availability of a bisphosphonate in autogenous free bone grafts. Bisphosphonate (0.01 mg/kg/day) was administered daily after an autogenous free bone graft on a rat calvarium. The effects of a bisphosphonate on the resorption of grafted bone and mRNA expression in bone specific genes, i.e. bone morphogenetic protein 2, bone morphogenetic protein 4, alkaline phosphatase, osteocalcin, osteoclast inhibitory factor and calcitonin receptor, were studied via a reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR and tartrate-resistant alkaline phosphatase (TRAP) staining. In a clinical and histomorphological review, bone resorption decreased in the experimental group in contrast to the control group where active bone resorption was observed. Bisphosphonate altered not only the mRNA expression of the bone resorption associated genes but also the bone formation associated genes. The expression of the calcitonin receptor (CTR) mRNA was not detected and the osteoclast inhibitory factor (OCIF) was significantly up-regulated in the experimental group as opposed to the control group. The expressions of osteocalcin and alkaline phosphatase mRNAs were also higher in the experimental group. However, there was no significant difference in the mRNA expression of bone morphogenetic proteins between the two groups. The data suggest the possibility of a clinical application of bisphosphonates for decreasing resorption of grafted bone.

The pathophysiology of adynamic bone disease

Adynamic bone disease was first noted in the early 1980s. It now represents the predominant bone lesion in peritoneal dialysis patients and is nearly as prevalent as osteitis fibrosis in hemodialysis patients. Bone turnover is markedly reduced in this disorder but, A in contrast to osteomalacia, there is no increase in osteoid formation. Although the pathophysiology of adynamic bone disease is still obscure, skeletal resistance to the calcemic action of PTH is important factor. Recently several study, whose reveal the down regulation of PTH receptor, osteoclast inhibitory factor and a assay of PTH, whole PTH play a role on the pathophysiology of skeletal resistance, were reported.

ELISA Methodology for Detection of Modified Osteoprotegerin in Clinical Studies

Osteoprotegerin (OPG), also known as osteoclast inhibitory factor, is a soluble receptor of the tumor necrosis factor receptor superfamily. The protein is secreted as a covalent, disulfide-linked homodimer, which is the predominant extracellular form (1), and is expressed in multiple tissues (1)(2)(3). OPG-mediated pathways might have a role in osteoporosis (3)(4)(5)(6) because estrogen increases OPG gene expression (4)(5). OPG maintains the structure of healthy bone and inhibits osteoclast activation and differentiation (3)(7). In the vascular system, OPG inhibits pathological calcification in the media intima (3). OPG has been proposed for therapy of osteopenic disorders, such as postmenopausal osteoporosis, Paget disease, rheumatoid arthritis, hypercalcemia, and lytic bone metastases (8). Initially, we developed an antibody-based ELISA method with an anti-human OPG monoclonal antibody for capture and an anti-human OPG polyclonal antibody for detection. Yano et al. (9) raised the concern for us that we may not detect the active dimeric OPG with antibody capture because they reported that serum OPG increased with age and that the monomer was the predominant form of OPG in human serum. Although they used a different antibody-dependent ELISA method (monoclonal capture and detection), the results reported by Yano et al. (9) do not correspond with the work performed at Amgen (1)(3)(4)(5)(7)(8)(10)(11)(12). We reasoned that OPG ligand (OPGL) (2)(7)(8)(10)(11)(12)(13)(14)(15)(16), also …