Immunological study on circulating murine osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF): possible role of OPG/OCIF in the prevention of osteoporosis in pregnancy

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family and plays a crucial role in the negative regulation of osteoclastic bone resorption. We have immunized OPG/OCIF knockout mice with murine rOPG/rOCIF and established a panel of hybridomas producing monoclonal antibodies (mAbs) to murine rOPG/rOCIF. Utilizing the mAbs, we developed enzyme-linked immunosorbent assay (ELISA) systems: one detecting both homodimeric and monomeric forms of murine OPG/OCIF and the other detecting only dimeric form of murine OPG/OCIF. With the aid of these ELISA systems we showed that OPG/OCIF is present mainly as a monomer in murine blood. The concentration of OPG/OCIF in normal mouse sera was approximately 500 pg/ml and there was no statistical difference in the serum concentration of OPG/OCIF among genders, age, and strains. Interestingly, the concentration of circulating OPG/OCIF in mouse markedly increased during pregnancy. The result indicated that circulating OPG/OCIF plays an important role in the protection of bone from excess resorption during pregnancy in mammals.     

A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function

Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family "osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)" as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.     

Reciprocal gene expression of osteoclastogenesis inhibitory factor and osteoclast differentiation factor regulates osteoclast formation

Osteoblasts/stromal cells support the formation of osteoclast-like cells (OCL) from osteoclast progenitor cells via expressing a membrane-associated protein, osteoclast differentiation factor (ODF), in the presence of osteotropic factors, whereas the cells secrete a substantial amount of osteoclastogenesis inhibitory factor (OCIF) in the unstimulated state. There are both OCL formation-supporting and the nonsupporting cell lines in osteoblasts/stromal cell lineages. The mechanism that divides osteoblasts/stromal cell lines into the two types is not known. The present study reports that OCL formation-supporting cell line ST2 showed a greatly increased level of ODF mRNA, whereas their OCIF mRNA was drastically diminished in the presence of 1alpha, 25(OH)2-dihydroxyvitamin D3 or prostaglandin E2. In contrast, MC3T3-E1 cells lacking OCL formation-supporting ability did not show a decrease in OCIF mRNA in response to the factors, despite a similar increase in ODF mRNA as ST2 cells. However, inactivated MC3T3-E1 cells secreting nothing supported OCL formation in coculture with human promyelocytic cells, HL60. On the contrary, ST2 cells did not support OCL formation from HL60 cells when cocultured in medium conditioned by 1alpha, 25(OH)2 vitamin D3-treated MC3T3-E1. These findings indicate that reciprocal gene expression of ODF and OCIF in osteoblasts/stromal cells is essential for supporting OCL formation.     

Interleukin-18 up-regulates osteoprotegerin expression in stromal/osteoblastic cells

Osteoprotegerin (OPG) and osteoclast differentiation factor (ODF) are crucial regulators of osteoclastogenesis. To determine the biological role of interleukin (IL)-18 produced by stromal/osteoblastic cells in osteoclastogenesis, we examined the effects of IL-18 on the OPG and ODF mRNA levels in these cells. When bone marrow stromal ST2 cells, osteoblastic MC3T3-E1 cells, and mouse calvarial osteoblasts were stimulated with IL-18, the expression of OPG mRNA, but not ODF mRNA, was transiently increased, its expression reaching a maximal level at 3 h after the beginning of the culture. In accordance with this observation, all these cells expressed the mRNAs of two IL-18 receptor components and MyD88, an adapter molecule involved in IL-18 signaling. Moreover, in these cells, mitogen-activated protein kinase was phosphorylated after stimulation with IL-18. These results suggest that stromal/osteoblastic cells are IL-18-responsive cells and that IL-18 may inhibit osteoclastogenesis by up-regulating OPG expression, without stimulation of ODF production, in stromal/osteoblastic cells.     

Low calcium environment effects osteoprotegerin ligand/osteoclast differentiation factor

In coculture with osteoblastic cell line MC3T3-E1 (E1) and mouse bone marrow cells, we reported that numbers of osteoclasts rose significantly on exposure to a low-calcium environment. Here we examined how osteoblasts influence osteoclastogenesis under a low-calcium environment. Comparing low extracellular calcium with a regular calcium environment, osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) mRNA expression show more increase in the culture of low-calcium environment than in that of a regular calcium environment. Calcium-sensing receptor (CaSR), which was supposed as one of the mechanisms of recognizing extracellular calcium, existedon the surface of E1 cells. When E1 cells stimulated with agonists of CaSR, gadolinium, and neomycin, OPGL/ODF mRNA expression decreased. Moreover, these agonists reduced osteoclast formation in coculture. Taken together, it is possible that osteoblasts may recognize extracellular calcium via CaSR and regulate osteoclastogenesis.     

Basic fibroblast growth factor inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) through suppressing the production of osteoclast differentiation factor.

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell (OCL) formation in cocultures of mouse spleen cells with either osteoblasts or a stromal cell line, ST2, in the presence of 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. bFGF directly acted on osteoblasts/stromal cells, but not osteoclast progenitors, to inhibit 1,25(OH)(2)D(3)-induced OCL formation. bFGF suppressed the mRNA expression of osteoclast differentiation factor (ODF) but did not affect that of osteoclastogenesis inhibitory factor (OCIF) in ST2 cells treated with 1,25(OH)(2)D(3) and dexamethasone. Enzyme-linked immunosorbent assay showed that bFGF hardly affected OCIF production in the treated ST2 cells. A genetically engineered soluble form of ODF, but not anti-OCIF neutralizing antibody, abolished bFGF-mediated inhibition of OCL formation. bFGF suppressed the binding of (125)I-labeled OCIF to both ST2 cells and osteoblasts treated with 1,25(OH)(2)D(3). These findings indicate that bFGF inhibits 1,25(OH)(2)D(3)-induced OCL formation via suppression of ODF production by osteoblasts/stromal cells.     

Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function.

Osteoclast differentiation factor (ODF), a novel member of the TNF ligand family, is expressed as a membrane-associated protein by osteoblasts/stromal cells. The soluble form of ODF (sODF) induces the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here, the effects of sODF on the survival, multinucleation, and pit-forming activity of murine osteoclasts were examined in comparison with those of M-CSF and IL-1. Osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts and bone marrow cells expressed mRNA of RANK (receptor activator of NF-kappaB), a receptor of ODF. The survival of OCLs was enhanced by the addition of each of sODF, M-CSF, and IL-1. sODF, as well as IL-1, activated NF-kappaB and c-Jun N-terminal protein kinase (JNK) in OCLs. Like M-CSF and IL-1, sODF stimulated the survival and multinucleation of prefusion osteoclasts (pOCs) isolated from the coculture. When pOCs were cultured on dentine slices, resorption pits were formed on the slices in the presence of either sODF or IL-1 but not in that of M-CSF. A soluble form of RANK as well as osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF, blocked OCL formation and prevented the survival, multinucleation, and pit-forming activity of pOCs induced by sODF. These results suggest that ODF regulates not only osteoclast differentiation but also osteoclast function in mice through the receptor RANK.     

Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells.

Basic fibroblast growth factor (bFGF) induced osteoclast formation in co-cultures of mouse spleen cells and osteoblasts. Osteoclastogenesis inhibitory factor (OCIF) and a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, abolished bFGF-induced osteoclast formation. bFGF did not affect spleen cells, but it did affect osteoblasts, to stimulate osteoclast formation. Northern blot analysis revealed that bFGF up-regulated the expression of osteoclast differentiation factor (ODF) and COX-2 and down-regulated the expression of OCIF in primary osteoblastic cells. NS-398 abolished the increase of ODF mRNA, but it had no effect on the decrease of OCIF mRNA. NS-398 suppressed the binding of (125)I-labeled OCIF to osteoblastic cells treated with bFGF. Enzyme-linked immunosorbent assay showed that bFGF inhibited OCIF production by osteoblastic cells, and the inhibition was not affected by NS-398. We conclude that bFGF induces osteoclast formation by stimulating ODF production through COX-2-mediated prostaglandin synthesis and by suppressing OCIF production through a mechanism independent of prostaglandin synthesis.     

Porphyromonas gingivalis-derived Lysine Gingipain Enhances Osteoclast Differentiation Induced by Tumor Necrosis Factor-α and Interleukin-1β but Suppresses That by Interleukin-17A: IMPORTANCE OF PROTEOLYTIC DEGRADATION OF OSTEOPROTEGERIN BY LYSINE GINGIPAIN*

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D₃-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159–166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.     

Connection between B lymphocyte and osteoclast differentiation pathways

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.     

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.     

Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

Osteoprotegerin and inflammation

RANK, RANKL, and OPG have well established regulatory effects on bone metabolism. RANK is expressed at very high levels on osteoclastic precursors and on mature osteoclasts, and is required for differentiation and activation of the osteoclast. The ligand, RANKL binds to its receptor RANK to induce bone resorption. RANKL is a transmembrane protein expressed in various cells type and particularly on osteoblast and activated T cells. RANKL can be cleaved and the soluble form is active. Osteoprotegerin decoy receptor (OPG), a member of the TNF receptor family expressed by osteoblasts, strongly inhibits bone resorption by binding with high affinity to its ligand RANKL, thereby preventing RANKL from engaging its receptor RANK. This system is regulated by the calciotropic hormones. Conversely, the effects of RANKL, RANK, and OPG on inflammatory processes, most notably on the bone resorption associated with inflammation, remain to be defined. The RANK system seems to play a major role in modulating the immune system. Activated T cells express RANKL messenger RNA, and knock-out mice for RANKL acquire severe immunological abnormalities and osteopetrosis. RANKL secretion by activated T cells can induce osteoclastogenesis. These mechanisms are enhanced by cytokines such as TNF-alpha, IL-1, and IL-17, which promote both inflammation and bone resorption. Conversely, this system is blocked by OPG, IL-4, and IL-10, which inhibit both inflammation and osteoclastogenesis. These data may explain part of the abnormal phenomena in diseases such as rheumatoid arthritis characterized by both inflammation and destruction. Activated T cells within the rheumatoid synovium express RANKL. Synovial cells are capable of differentiating to osteoclast-like cells under some conditions, including culturing with M-CSF and RANKL. This suggests that the bone erosion seen in rheumatoid arthritis may result from RANKL/RANK system activation by activated T cells. This opens up the possibility that OPG may have therapeutic effects mediated by blockade of the RANKL/RANK system.     

Characterization of synovial cell clones isolated from rheumatoid arthritis patients: possible involvement of TNF-alpha in reduction of osteoprotegerin in synovium

To elucidate the role of the synovium in bone destruction by osteoclasts in rheumatoid arthritis (RA), primary synovial cells isolated from RA patients were cultured and characterized. The cultured primary cells did not produce RANKL (TRANCE/ODF/OPGL/TNFSF11/CD254), an inducer of osteoclast differentiation, but constitutively produced its inhibitor, osteoprotegerin (OPG). Addition of TNF-alpha to the primary cultures of synovial cells reduced the cell viability and strongly suppressed OPG production. We then established nine synovial cell clones, including SYM-1, responsible for OPG production from primary synovial cell cultures. TNF-alpha induced apoptosis of SYM-1 cells within 24h and decreased OPG levels, while infliximab, a chimerical form of the anti-TNF-alpha antibody drug, suppressed the apoptosis and restored OPG levels. These results suggest the existence of fibroblastic cells producing OPG in the synovium, while TNF-alpha suppresses OPG production by inducing apoptosis in those cells. Further, infliximab is considered to inhibit bone destruction through restoration of OPG levels in RA.     

Prostaglandin E receptor EP4 antagonist suppresses osteolysis due to bone metastasis of mouse malignant melanoma cells

We examined the effects of prostaglandin E (PGE) receptor subtype EP4 antagonist on bone metastasis of cancer to clarify PGE's role in bone metastasis. Metastatic regions were detected in femurs accompanying severe bone loss in mice injected with B16 malignant melanoma cells. Administration of EP4 antagonist restored the bone loss induced by B16 melanoma. Adding B16 cells induced osteoclast formation in the coculture of bone marrow cells and osteoblasts without any exogenous bone-resorbing factor, and EP4 antagonist completely suppressed the osteoclast formation induced by B16 cells. Therefore, EP4 antagonist is a possible candidate for the therapy of bone metastasis of cancer.     

Human periodontal ligament cells derived from deciduous teeth induce osteoclastogenesis in vitro

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins involved in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament cells derived from deciduous teeth (DPDL cells) and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated by application of 10(-8) M 1 alpha, 25(OH)2 vitamin D3 [1,25-(OH)2D3] and 10(-7) M dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated a decrease in OPG following application of 1, 25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) were induced when DPDL cells were co-cultured with mouse bone marrow cells in the presence of 1,25-(OH)2D3 and Dex. TRAP-positive MNCs increased significantly when the DPDL cells were co-cultured with bone marrow cells in the presence of anti-human OPG antibody together with 1, 25-(OH)2D3 and Dex. These results indicate that PDL cells derived from deciduous teeth synthesize both RANKL and OPG and could regulate the differentiation of osteoclasts.     

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Osteolytic Breast Cancer Bone Metastasis and Estrogen Deficiency-Induced Bone Loss

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ₂ on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ₂ dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ₂ suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ₂ blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ₂ exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ₂ substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ₂ prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ₂ may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.